Print version ISSN 0104-6632
Braz. J. Chem. Eng. vol.20 no.1 São Paulo Jan./Mar. 2003
Affinity chromatography with pseudobiospecific ligands on high-performance supports for purification of proteins of biotechnological interest
N.B.IannucciI; F.J.WolmanI; S.A.CamperiI; A.A.N.CañizoI; M.GrasselliII; O.CasconeI
ICátedra de Microbiología Industrial y Biotecnología, Facultad de Farmacia y Bioquímica , UBA Junín 956, 1113, Buenos Aires, Argentina
IIDepartamento de Ciencia y Tecnología, Universidad Nacional de Quilmes, Argentina
High-performance affinity matrices were obtained by attaching pseudobiospecific ligands to hollow-fibre membranes. The neutral protease contained in FlavourzymeTM was purified to homogeneity with Yellow 4R-HE affinity hollow-fibre membranes. Immobilisation of Red HE-3B allowed purification of a milk-clotting enzyme obtained by solid-state culture of Mucor bacilliformis. Copper immobilisation through iminodiacetic acid allowed fractionation of Biocon Bioconcentrated PlusTM to separate the pectinesterase-containing fraction. The productivity of the developed processes - 1900, 94 and 750 U/ml.min, respectively was 10- to 15-fold higher than that achieved with the same ligands immobilised on agarose-based soft gels, mainly due to the shortening of the purification processes.
Keywords: affinity chromatography, pseudobiospecific ligands, protein purification.
Affinity chromatography is now a suitable technique for industrial protein purification processes, with pseudobiospecific ligands largely responsible for this success due to their low cost, availability, simple immobilisation reaction, resistance to chemical and biological degradation as well as acceptable selectivity and capacity (Lowe and Pearson, 1984). Triazine dyes and immobilised metal ions are good examples of these ligands.
Immobilised metal ion affinity chromatography (IMAC) is a protein purification method that takes full advantage of metal ion affinities of surface functional groups, mainly histidines (Porath et al., 1975). Triazine dyes have also been utilised to obtain affinity chromatography matrices. In early studies dyes were used almost exclusively to purify dehydrogenases and kinases because of their affinity for these enzymes. However, as triazine dyes contain polar and non-polar groups, they can interact rather selectively with other proteins by both hydrophobic and electrostatic forces (Lowe and Pearson 1984).
Pseudobiospecific ligands have been immobilised on a wide variety of support matrices in the search for an ideal system. Some of the supports examined in this way include agarose, dextrans, polyacrylamide, agarose-polyacrylamide copolymers, cellulose and glass. Of these, soft gels have been extensively used as support matrices for pseudo-biospecific ligands (Angal and Dean, 1977; Camperi et al., 1996).
Microfiltration affinity membranes are a good alternative to macroporous affinity beads as proteins are directly transported by convection to the affinity group onto the inner surface of the microfiltration membrane, thus making adsorption rates faster. Additionally, membrane chromatography can overcome the high operating pressure characteristic of bead-based chromatography (Brandt et al., 1988; Gebauer et al., 1997). Moreover, solutions containing debris or solid particles can be processed directly in the cross-flow mode (Camperi et al., 1999). The hollow-fibre membrane has an advantage over the flat-sheet membrane: the scale-up can be achieved by simple bundling of a number of hollow fibres - something suitable for scale production and process requirements (Kubota et al., 1997).
The aim of this work was to demonstrate the usefulness of chromatographic matrices obtained by attaching pseudobiospecific ligands to high-performance supports for industrial enzyme purification, using proteases and pectinases as model enzymes.
MATERIALS AND METHODS
The PD-10 columns were from Amersham Pharmacia Biotech, Uppsala, Sweden. The dyes, azocasein and citric pectin were from Sigma-Aldrich, St. Louis, USA. FlavourzymeTM was from Novo Industries, Denmark. The Bioconcentrated PlusTM was from Biocon, Ireland. The extracts containing M. bacilliformis acid protease were prepared according to Fernández Lahore et al. (1998). All other reagents were AR grade.
Preparation of Affinity Hollow-Fibre Membranes
Membranes containing Cibacron Blue F3G-A, Red HE-3B and Yellow 4R-HE for affinity chromatography were prepared according to Wolman et al. (2000) using polyethylene hollow-fibre microfiltration membranes, donated by Dr. F. Yoshii (Takasaki Radiation Chemistry Research Establishment, Japan). They had a nominal 0.33 µm internal pore diameter and nominal 70% porosity. Inner and outer diameters were 0.6 and 1.2 mm, respectively. IDA membranes were prepared from polysulfone hollow-fibre microfiltration membranes, a gift of A/G Technology Co., Needham, Massachusetts, USA, as described by Camperi et al. (2000). They had a nominal 0.65 µm internal pore diameter and nominal 80% porosity. The inner and outer diameters were 0.75 and 1.25 mm, respectively.
Pectinlyase (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.
Pectinesterase (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.
Neutral protease assay was carried out by the method of Charney and Tomarelli (1949), modified by Vázquez et al. (2000). One protease unit is defined as the amount of enzyme that causes a rise in absorbance of 0.003 per min at 340 nm.
Milk-clotting activity was determined as described by Arima et al. (1970). The unit of milk-clotting activity (CU) is defined as the amount of enzyme capable of clotting 1.0 ml of substrate in 40 min at 35°C.
Total Protein Measurement
It was carried out by the method of Bradford (1976).
SDS-Polyacrylamide Gel Electrophoresis
It was performed in Phast System equipment (Amersham Pharmacia Biotech, Uppsala, Sweden) as per the instructions of the manufacturer.
Adsorption Isotherm Measurement
Adsorption isotherms for neutral protease from FlavourzymeTM, acid protease from M. bacilliformis solid-state culture extract and pectinesterase from Biocon Bioconcentrated PlusTM were measured basically as described by Chase (1984). A 20 mM sodium acetate buffer, pH 5.0; a 50 mM sodium acetate buffer, pH 4.1 and a 20 mM sodium phosphate buffer, pH 7.0, 0.25 M NaCl, were used as the adsorption buffers, respectively. Small pieces of affinity membrane were put into tubes (approximately 10 µl membrane volume in each one) containing increasing amounts of each adsorbate to obtain 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 supernatant solution was then removed and its enzyme activity at equilibrium was determined as indicated above. The equilibrium activity of the enzyme bound to the membrane per unit of membrane volume was calculated as the amount present at the beginning of the experiment less the amount still in soluble phase at equilibrium.
Dissociation constant (Kd) and maximum capacity (qm) values were determined according to Chase (1984).
Enzyme Purification Process
Enzymes were purified by loading the sample solution to a cartridge assembled with 10 affinity hollow-fibre membranes, 8 cm long (membrane volume = 0.408 ml). After saturation and washing with 5 membrane volumes of adsorption buffer, proteases were eluted with 1 M NaCl in the adsorption buffer and pectinesterase with 20 mM sodium phosphate buffer, 0.1 M EDTA, pH 7.0. The cartridge was then regenerated with NaOH 0.1 M. Spatial velocity (flow rate divided by the membrane volume) was 20 min-1.
RESULTS AND DISCUSSION
Previous work with beaded chromatography matrices allows us to select Yellow 4R-HE, Red HE-3B and Cibacron Blue F3G-A as suitable ligands for the neutral protease from Flavourzyme, Red HE-3B for the milk-clotting enzyme from M. bacilliformis and IDA-Cu for pectinesterase from Biocon Bioconcentrated Plus (Camperi et al., 1996; Navarro del Cañizo et al., 2000 and 2001).
Neutral Protease from Flavourzyme
Figure 1 shows the isotherms for adsorption of the neutral protease contained in Flavourzyme on dye-affinity membranes at pH 5.0. From these isotherms, qms of 19090, 12930 and 13870 U/ml for Yellow 4R-HE, Red HE-3B and Cibacron Blue F3G-A, respectively, were calculated. Kds were 18, 15 and 17 U/ml, respectively.
In developing a protease purification process, different eluents were assayed. Ethyleneglycol and isopropanol only eluted less than 5% of adsorbed protease, while 1 M NaCl in 20 mM sodium acetate buffer, pH 5.0, eluted more than 90% of adsorbed protease on the three dye membranes, thus providing evidence that binding forces are mainly electrostatic. Yellow 4R-HE membranes were selected for productivity studies on the basis of their higher capacity.
When the dye membrane was fed with a 2 mg/ml Flavourzyme solution in 20 mM sodium acetate buffer, pH 5.0, and after a washing step, the protease was eluted with 1M NaCl in the adsorption buffer, 89% of electrophoretically pure enzyme was recovered. The productivity of the process was 1900 U/ml.min, 10 times higher than that achieved with beaded dye-affinity matrices.
Acid Protease from M. bacilliformis
When the clarified aqueous crude extract of the M. bacilliformis solid-state culture was set to pH 4.1, the protease was not adsorbed on Red HE-3B. A similar interference was reported for ion-exchange purification of the protease (Fernández Lahore et al., 1998). However, after conditioning of the crude extract by size-exclusion chromatography on a PD-10 column equilibrated in 50 mM sodium acetate buffer, pH 4.1, the acid protease bound to the immobilised dye. A qm of 950 U/ml and a Kd of 1.3 U/ml can be calculated from the corresponding isotherm (not shown).
As in the case of the neutral protease from Flavourzyme, 1 M NaCl in the adsorption buffer was more efficient than hydrophobic eluents, thus providing evidence of the electrostatic nature of the ligand-acid protease bond.
After the purification process in a Red HE-3B hollow-fibre cartridge, eluted protease yielded a single band in SDS-PAGE, thus offering evidence of the usefulness of the method proposed. The productivity of the process was 94 U/ml.min, and 92% of the milk-clotting enzyme could be recovered.
Pectinesterase from Biocon Bioconcentrated Plus
With Cu2+ as the immobilised metal and 20 mM sodium phosphate, pH 7.0, 0.25M NaCl, as the adsorption buffer, PE was fully retained by the chromatographic matrix while the fraction not retained contained all the PL of the commercial preparation. From the equilibrium adsorption isotherm, the maximum capacity for PE under the above conditions was 8000 U/ml and the Kd value, 20.3 U/ml.
In order to test the usefulness of the cartridge for pectic enzyme fractionation, the IDA hollow-fibre cartridge was loaded with 3000 U of PE and 1445 U of PL (5 ml of Biocon Bioconcentrated Plus 23 mg/ml). Figure 2 shows the pattern obtained. Ninety-nine per cent of the PE activity was retained by the chromatographic matrix and eluted quantitatively with 20 mM sodium phosphate buffer, 0.1 M EDTA, pH 7.0, thus indicating that the fractionation procedure can be successfully scaled up. The time of the fractionation process was far shorter than when working with chelating soft gel (Camperi et al., 1996), where lower flow rates must be used to allow mass transfer. The better hydrodynamic properties of the membranes resulted in enormous savings of time and a higher productivity: 750 PE U/ml.min compared with that previously obtained working with chelating soft gels, 52 U/ml.min (Camperi et al., 1996).
The usefulness of chromatographic matrices, obtained by attaching pseudobiospecific ligands to hollow-fibre membranes, for purification of neutral and acid proteases and fractionation of pectinases was demonstrated.
The high capacity of the membrane cartridge and its excellent hydrodynamic properties allow a very fast fractionation of commercial enzyme preparations at a low operating pressure, thus resulting in purification processes with productivities 10 to 15 times higher than those with conventional beaded matrices.
This work was supported by grants from the Universidad de Buenos Aires, Agencia Nacional de Promoción Científica y Tecnológica de la República Argentina and Consejo Nacional de Investigaciones Científicas y Técnicas de la República Argentina. SAC, MG and OC are career researchers at the CONICET.
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Address to correspondence
Received: March 5, 2002
Accepted: September 12, 2002