Engineering and Manufacturing for Biotechnology - Marcel Hofman & Philippe Thonart

Separation of -lactalbumin and -lactoglobulin by preparative chromatography

and -lactalbumin proteins have different affinities to the ionic exchange resin in the experimental conditions tested, as can be observed in Figure 3. Thus, as -lactoglobulin has higher affinity to the resin than -lactalbumin it should be expected when a mixture of these two proteins are been separated in a SMB that -lactoglobulin will be present at the extract stream. In order to examine the performance of the SMB to separate mixtures of -lactoglobulin and -lactalbumin, equations (2) to (5) and (9) to (10) were simulated. The dynamic values of the proteins concentration in the extract and raffmate obtained by simulation can be observed in Figure 5. In the conditions tested, the steady state is obtained after 180 minutes and the products in the raffinate and in the extract are practically pure. The -lactoglobulin is recovered preferentially in the extract and is obtained in more diluted form than the -lactalbumin due to their higher affinity to the resin that implies in a higher desorbant flow rate to remove them to the solid phase. We can also notice in Figure 5 that the protein concentrations in the exit streams of the

SMB are dependent to the switching time.

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S.L. Lucena, P.T.V. Rosa, L.T. Furlan and C.C. Santana

At the time that the SMB reaches the steady state, there will be a well-established concentration profile inside the columns. Figure 6 shows the stationary concentration

by the switching time. In order to verify the influence of this parameter on the separation of -lactoglobulin and -lactalbumin, the equations (2) to (5) and (9) to (10) were simulated for several values of switching time keeping constant the values of the flow rates in the sections. The results obtained by simulation are presented in Figure 7. We can observe in this figure that there is a range of switching time values (from 11.75 to 13.50 minutes) where the raffinate and the extract are completely pure. For switching times lower than 11.75 minutes, the -lactalbumin is not completely removed from the solid phase in the section III and will be eluted at section IV, contaminating the extract.

For switching times greater than 13.50 minutes, there is some desorption of lactoglobulin from section II that will contaminate the raffinate. The range of switching time were the separation is complete is not the same where the concentrations of the compounds in the exit streams are at the maximum values. Thus, someone should decide if purity or concentration is the main purpose of the separation in order to set the switching time.

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Separation of -lactalbumin and -lactoglobulin by preparative chromatography

6. Conclusions

Adsorption isotherms for the proteins -lactalbumin and -lactoglobulin were experimentally obtained. The developed mathematical models are capable to predict the breakthrough curves for adsorption columns operating separately for three types of

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isotherms of adsorption. The mathematical model developed for the simulated moving bed represents the performance of the system on separating individual components from a mixed feed. The computational routine for the simulated moving bed for compositions was solved for the case of an isolated adsorption column, as presented in the Figure 4. The parameters of the isotherms experimentally obtained indicates that the proteins are strongly adsorbed in the studied conditions (high values of what would imply in a very slow movement of the proteins along the adsorbed system. The parameters of adsorption isotherms can be modified through the increase of the ionic force or of the pH of the media, turning possible the use of this technique for the purification of those proteins.

References

Barker, P.E. and Abusabah, E.K.E. (1985), The separation of Synthetic Mixtures of Glucose and Fructose and also Inverted Sucrose Feedstocks Using Countercurrent Chromatographic Techniques,

Chromatographia, vol. 20, no. 1, 9-12.

Beszedits, S. (1982), Protein recovery from Food Processing Wastewater, B&L Information Services. Blanch, II.B. and Clark, D. S. (1997), Biochemical Engineering, Chapter 6: Product Recovery, Marcel

Dekker Inc., New York.

Carrére, H., Extraction des Proteines du Lactoserum par Chromatographie d' Èchange d' ions en Lit Fluidisé, Thése de Doctorat, Institute Polytechnique de Toulouse, 1993.

Dankwerts, P.V., Chemical Engineering Science, (1953), 2, 1-12 .

Ganetsos, G. and Barker, P.E., Eds. (1994), Preparative and Production Scale Chromatography, Marcel

Dekker, Inc., New York.

Gottschlich, N., Weidgen, S., Kasche, V. (1996). Continuous biospecific affinity purification of enzymes by simulated moving-bed chromatography: theoretical description and experimental results, Journal of Chromatography ,719, 267-274.

Gottschlich, N. and Kasche, V., (1997). Purification of monoclonal antibodies by simulated moving-bed chromatography, Journal of Chromatography , 765, 201-206.

Huang, S.Y., Lin, C.K., Chang, W.H. and Lee, W.S., (1986) Enzyme purification and concentration by simulated moving bed chromatography: in the experimental study, Chemical Engineering Communications, Vol. 45, 291-309

Press, W . H, Teukolsky, S.A., Vetterling, W.T. and FlannerY, B.P. (1992), Numerical Recipes in Fortran, Cambridge University Press.

Yamamoto, S., Nakanishi, K. It is Matsuno, R. (1988),. Ion Exchange Chromatography of Proteins, Marcel

Dekker, Inc., New York.

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Separation of -lactalbumin and ß-lactoglobulin by preparative chromatography

Appendix

Dimensionless forms of Equation (1) for several isotherms

Linear Isotherm

Langmuir Isotherm

Langmuir Competitive Isotherm

where :

In the above equations the dimensionless parameters:

TN is an arbitrary time, L is the length of the column, K is the constant of the linear isotherm, it association constant and is the maximum adsorption capacity of the resin

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HIGH-SPEED PECTIC ENZYME FRACTIONATION BY IMMOBILISED METAL ION AFFINITY MEMBRANES

SILVIA ANDREA CAMPERI, MARIANO GRASSELLI1 AND OSVALDO CASCONE

Cátedra de Microbiología Industrial y Biotecnologia. Facultad de Farmacia y Bioquimica. UBA. (1113) Junín 956, Buenos Aires,

Argentina.

1Departamento de Ciencia y Tecnologia. Universidad Nacional de

Quilmes. Roque Sáenz Peña 180, (1876) Bernal, Prov. Buenos Aires, Argentina. E-mail: scamperi@ffyb.uba.arFax: 54-11-4508-3645

Abstract

Immobilised metal ion affinity polysulphone hollow-fibre membranes with a high capacity for protein adsorption were prepared and their application for commercial pectic enzyme fractionation was studied. The pass-through fraction containing pectin lyase (PL) is useful for fruit-juice clarification without methanol production on account of pectin-esterase (PE) being retained by the membrane.

1. Introduction

Commercial preparations of pectic enzymes normally contain a mixture of depolymerising (pectin lyase, PL, and polygalacturonase, PG) and de-esterifying (pectinesterase, PE) enzymes (Rombouts and Pilnik, 1980).

The use of PL alone, instead of the combination of PE and PG for fruit juice clarification, prevents the release of methanol in the juice, thus constituting a potential health hazard in non-concentrated juices (Szajer and Szajer, 1982). Moreover, the volatile ester content, responsible for the specific aroma of various fruits, is not damaged (Alaña et al., 1989). Furthermore, the use of PG and PE -containing enzyme complexes decreases fruit juice stability because of the coagulating processes caused by the interaction of the de-esterified pectin derivatives with the endogenous For these reasons and in order to utilise pectinase activities more rationally, there is a current need for purification of commercial pectinase preparations to allow more specific and controllable effects (Alaña et al., 1989).

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M. Hofman and P. Thonart (eds.), Engineering and Manufacturing for Biotechnology, 339–349.

© 2001 Kluwer Academic Publishers. Printed in the Netherlands.

Silvia Andrea Camperi,_Mariano Grasselli and Osvaldo Cascone

We reported a fractionation method to separate PL from PE and PG to obtain a fraction which will not produce methanol during juice clarification (Navarro del Cañizo et. al., 1994, Camperi et al., 1996). This is based on immobilised metal ion affinity chromatography (IMAC ), a protein purification method that exploits the affinity of surface functional groups, mainly histidines, towards transition metals (Porath et al., 1975, Hemdan et al., 1989). IMAC is a good option in preparative protein purification, taking into account its high yields, and ligand economy and stability (Arnold, 1991). Due to beaded soft gels are utilised as supports, the main drawback of this fractionation scheme is that low flow rates must be used to prevent gel deformation and allow mass transfer. Also, the sample must be clarified before loading onto the column.

Membrane (internal pore diameter between 0.1 and 1 µ m) is a good alternative to macroporous beads as separation based on membranes is characterised by the absence of

the affinity ligand onto the inner surface of the through-pores of the membrane thus making adsorption rates faster. Additionally, membrane chromatography can overcome the high operating pressure and low adsorption rate, the typical disadvantages of bead- based chromatography (Brandt et al., 1988; Roper and Lightfoot, 1995; Thömmes and Kula, 1995). Furthermore, it has been proposed that, unlike conventional column chromatography, solutions containing debris or solid particles can be processed by crossflow microfiltration in chromatographic membranes without a previous clarification treatment (Kroner et al., 1992).

Saito et al. (1989) developed a new type of affinity hollow-fibre membrane by radiation-induced co-grafting of a cross-linking agent with the reactive monomer. Grafting is a useful method for chemical modification of existing polymers. In this way, a higher degree of chemical modification of chromatographic supports can be obtained, thus meaning a greater amount of reacting sites for ligand attachment to the support (Mueller-

Schulte and Daschek, 1995).

A number of different module configurations (hollow fibres, spiral-wound cartridges, flat-sheet membranes, etc.) are available on the market. A hollow-fibre membrane is superior to a flat-sheet membrane because of its high surface area/volume ratio (Yamagishi et al. 1991) and the easy scale-up of the chromatography by simple bundling of numbers of hollow fibres (Kubota et al., 1997).

Polysulphone is a suitable membrane polymer because of its good film-forming properties and its resistance to thermal and biological degradation. Its heat stability allows performing chemical modifications without impairing its performance. We made tentacle cation-exchange hollow-fiber membranes of high capacity for proteins from epoxy- activated microfiltration polysulphone membranes (Camperi et al., 1999).

Here we report a simple and economical method for pectinase fractionation, based on affinity chromatography, using a cartridge of Cu(II)-iminodiacetic (IDA) as the immobilised ligand on polysulphone membranes.

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2. Materials and methods

2.1. ENZYMES AND REAGENTS

Polysulphone hollow-fibre microfiltration membranes, kindly donated by A/G Technology Co., Needham, Massachusetts, USA were epoxy-activated by Innovatec S.A., Buenos Aires, Argentina. They had a nominal 0.65 µm internal pore diameter and a nominal 80% porosity. The inner and outer diameters were 0.75 and 1.25 mm respectively.

Chicken egg lysozyme, horse skeletal muscle myoglobin, haemoglobin and citric pectin were from Sigma Chemical Co., St. Louis, USA. L-histidine hydrochloride was from BDH Chemicals Ltd., England. Bioconcentrated Plus, Biocon, Ireland was utilised as a pectic enzyme source.

All other reagents were AR grade.

2.2. HISTIDINE, LYSOZYME, MYOGLOBIN AND HAEMOGLOBIN CONCENTRATION MEASUREMENTS

Histidine solution concentration was determined by measuring their absorbance at 220 nm, lysozyme and myoglobin at 280 nm, and haemoglobin at 430 nm.

2.3. PECTIC ENZYME ASSAY

PL was assayed by monitoring the increase in absorbance at 235 nm as described by Albersheim (1966). One PL unit is defined as the amount of enzyme that causes a rise in absorbance of 1.0 per min, at 235 nm.

PE activity was determined by monitoring the decrease in absorbance of bromocresol green at 617 nm due to carboxyl groups being released in pectin according to Vilariño et al. (1993). One PE unit is the amount of enzyme required to release 1 µEq of carboxyl groups per min.

2.4. CHELATING HOLLOW FIBRE SYNTHESIS

Iminodiacetic acid (IDA) was immobilised onto the epoxy-activated membranes by suspending the fibres in 1M IDA-2Na in dimethyl sulphoxide (DMSO) water (1:1) (Yamagishi et al. 1991). The reaction was performed at 80°C for 24 h. In order to hydrolyse the remaining epoxy groups, the fibres were then immersed in 0.5 M sulphuric acid for 2 h at 80°C. After washing the fibres with water, they were immersed in 0.5 Three hours later they were washed again with distilled water. Figure 1 shows a scheme of the chelating hollow-fiber membranes.

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2.5. PURE WATER FLUX DETERMINATION FOR A SINGLE CHELATING HOLLOW FIBRE.

It was determined with a dead-end constant pressure apparatus as described by Yamagishi et al. (1991). An 8-cm long hollow fibre was positioned in a U-shaped configuration and pure water was forced to permeate outwards at a constant filtration pressure of 1 bar, in the dead-end mode. Space velocity (SV) was calculated as the flow rate divided by the membrane volume.

2.6. MEASUREMENT OF THE AMOUNT OF IDA INTRODUCED

The amount of IDA introduced was determined from the measurement of the copper saturation capacity assuming a stochiometric ratio. Copper content was measured spectrophotometrically by soaking the fibres with 0.1 M EDTA, pH 7.5, for 3 h and

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comparing the absorbance of the supernatant at 715 nm with that of 0.1 M EDTA with Cu(II) at various concentrations (Wuenschell et al., 1991).

2.7. ADSORPTION ISOTHERMS MEASUREMENT

The adsorption isotherms for histidine, lysozyme, myoglobin, haemoglobin and pectinesterase binding to IDA membranes were measured basically as described by Chase (1984). A 20 mM sodium phosphate buffer, pH 7.0, 0.25 M NaCl was used as the adsorption buffer. Small pieces of IDA membrane were put into tubes (approximately

10 ul membrane volume in each one) containing increasing amounts of each adsorbate, in a final volume of 1.5 ml. The suspension was stirred gently for 24 h at 20°C to allow the system to reach its equilibrium. Each adsorbate solution was then removed and its concentration or activity at equilibrium (c*) was determined as indicated above. The equilibrium concentration or activity of adsorbate bound to the membrane per unit of total membrane volume (q*), was calculated as the total amount of adsorbate present at the beginning of the experiment less the amount still in the soluble phase at equilibrium.

Values for the dissociation constant (Kd) and the maximum adsorption capacity (qm) were determined according to Chase (1984) and are given as the mean

2.8. ASSEMBLING A HOLLOW-FIBRE MEMBRANE MODULE

A/G Technology Co., Needham, Massachusetts, USA donated the module cartridge.

The cartridge had four openings: two on the lumen side and two on the shell side. Ten chelating hollow fibres, 8 cm long, were put into the cartridge in parallel and plugged at both ends using epoxy resin. The effective membrane length was 6.5 cm (total volume, 0.408 ml).

2.9. BREAKTHROUGH CURVES FOR PE AND PL ADSORPTION

The sample was a solution of Biocon Bioconcentrated Plus 23 mg/ml in a 20 mM sodium phosphate buffer, pH 7.0, 0.25 M NaCl containing 600.3 U/ml of PE and 289 U/ml of PL. It was pumped at a SV of 5 through the cartridge in the dead-end flow mode. The lumen side was used as an inlet and the shell side as an outlet for the permeate. The outlet of the cartridge was monitored for PE and PL activity in all the fractions collected. Figure 2 shows a schematic diagram of the system utilised for breakthrough curves measurement.

2.10. UTILISATION OF THE CU(II)IDA-CARTRIDGE FOR PECTIC ENZYME

FRACTIONATION

5 ml solution of Biocon Bioconcentrated Plus 23 mg/ml in 20 mM sodium phosphate buffer, pH 7.0, 250 mM NaCl was pumped through the cartridge in the dead-end flow mode. PE was eluted with 0.1 M EDTA, pH 7.0.

The activity of PE and PL was measured in the washing and in eluate solutions.

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