Purificação de três diferentes beta-galactosidades microbianas por partição em sistemas de duas fases aquosas

Purification of three different microbial beta-galactosidases by partitioning in aqueous two-phase systems

Resumos

Este trabalho tratou da investigação do efeito do peso molecular de polietilenoglicol (PEG) sobre a partição de enzimas beta-galactosidases de diferentes origens microbianas: Escherichia coli, Klueveromyces lactis e Aspergillus orizae em sistemas de duas fases aquosas (SDFA).Foi observado que os melhores sistemas para purificação da enzima de E. coli foram os formados por PEG 4000, 6000 e 8000/fosfato, fornecendo os mais elevados fatores de purificação da enzima. As enzimas de Klueveromyces lactis e Aspergillus orizae não foram eficientemente purificadas nestes sistemas sendo insensíveis à alterações do peso molecular do PEG. Portanto, um outro sistema de duas fases aquosas foi desenvolvido contendo um ligante específico, p-aminofenil 1-tio-beta-D-galactopiranosídeo (APGP), acoplado ao PEG para purificar a enzima de Klueveromyces lactis. Uma etapa simples de partição no SDFA formado por 6% APGP-PEG4000 + 12% dextrana T505.000 foi capaz de recuperar 83% da enzima na fase superior do sistema e de aumentar 1,6 vezes o fator de purificação.

beta-galactosidase; recuperação de proteína; sistemas aquosos bifásicos

This work investigated the effect of the molecular weight of polyethyleneglycol (PEG) upon the partition coefficient of beta-galactosidases from three different microorganisms: Escherichia coli, Klueveromyces lactis and Aspergillus orizae. It was found that PEG 6,000 and PEG 8,000/phosphate were the best systems for achieving the highest purification factors of E. coli beta-galactosidase. However, the other two yeast beta-galactosidases were not efficiently separated from their contaminants in any of the PEG/salt systems. In order to improve the separation of Klueveromyces lactis beta-galactosidase from the main protein contaminants, the biospecific ligand p-aminophenyl 1-thio-beta-D-galactopyranoside (APGP) was attached to activated PEG 4000. The affinity PEG having APGP bound to its backbone was synthesized in two steps. The partitioning of Klueveromyces lactis beta-galactosidase in aqueous two-phase systems prepared with 6% APGP-PEG4000 + 12% dextran T505,000 increased 1.6-fold the purification factor of the target enzyme, allowing the recovery of 83% of the enzyme in the top PEG-rich phase.

beta-galactosidase; protein recovery; aqueous two-phase systems; downstream processing

PURIFICATION OF THREE DIFFERENT MICROBIAL b-GALACTOSIDASES BY PARTITIONING IN AQUEOUS TWO-PHASE SYSTEMS¹

SILVA², Maria Estela; PELLOGIA², Cássia; PIZA², Francisco Assis Toledo & FRANCO²,^*, Telma Teixeira

SUMMARY

This work investigated the effect of the molecular weight of polyethyleneglycol (PEG) upon the partition coefficient of b-galactosidases from three different microorganisms: Escherichia coli, Klueveromyces lactis and Aspergillus orizae. It was found that PEG 6,000 and PEG 8,000/phosphate were the best systems for achieving the highest purification factors of E. coli b-galactosidase. However, the other two yeast b-galactosidases were not efficiently separated from their contaminants in any of the PEG/salt systems. In order to improve the separation of Klueveromyces lactis b-galactosidase from the main protein contaminants, the biospecific ligand p-aminophenyl 1-thio-b-D-galactopyranoside (APGP) was attached to activated PEG 4000. The affinity PEG having APGP bound to its backbone was synthesized in two steps. The partitioning of Klueveromyces lactis b-galactosidase in aqueous two-phase systems prepared with 6% APGP-PEG4000 + 12% dextran T505,000 increased 1.6-fold the purification factor of the target enzyme, allowing the recovery of 83% of the enzyme in the top PEG-rich phase.

Key-words: b-galactosidase, protein recovery, aqueous two-phase systems, downstream processing.

RESUMO

PURIFICAÇÃO DE TRÊS DIFERENTES b-GALACTOSIDASES MICROBIANAS POR PARTIÇÃO EM SISTEMAS DE DUAS FASES AQUOSAS. Este trabalho tratou da investigação do efeito do peso molecular de polietilenoglicol (PEG) sobre a partição de enzimas b-galactosidases de diferentes origens microbianas: Escherichia coli, Klueveromyces lactis e Aspergillus orizae em sistemas de duas fases aquosas (SDFA).Foi observado que os melhores sistemas para purificação da enzima de E. coli foram os formados por PEG 4000, 6000 e 8000/fosfato, fornecendo os mais elevados fatores de purificação da enzima. As enzimas de Klueveromyces lactis e Aspergillus orizae não foram eficientemente purificadas nestes sistemas sendo insensíveis à alterações do peso molecular do PEG. Portanto, um outro sistema de duas fases aquosas foi desenvolvido contendo um ligante específico, p-aminofenil 1-tio-b-D-galactopiranosídeo (APGP), acoplado ao PEG para purificar a enzima de Klueveromyces lactis. Uma etapa simples de partição no SDFA formado por 6% APGP-PEG4000 + 12% dextrana T505.000 foi capaz de recuperar 83% da enzima na fase superior do sistema e de aumentar 1,6 vezes o fator de purificação.

Palavras-chave: b-galactosidase, recuperação de proteína, sistemas aquosos bifásicos.

1 — INTRODUCTION

The enzymatic hydrolysis of lactose to glucose and galactose with b-galactosidase is one of the most ancient biotechnological processes known to humanity. The microorganisms usually associated with b-galactosidase high production are the Escherichia coli, Bacillus megaterium, Bacillus stearothermophillus bacteria and the Kluyveromyces lactis and Aspergillus orizae yeasts, in liquid (16) and in solid-state fermentation (3). The improvement of b-galactosidase downstream processing from its microbial sources would be desirable to reduce the cost of enzyme preparation, since extraction, purification and concentration steps are commonly responsible for up to 40% of total production cost (9).

Purification of target proteins requires their separation from the media or from the raw extract used for the maintenance of the biomolecules. The product is usually present at low levels and also needs to be concentrated. Protein extraction in aqueous two-phase systems (ATPS) is a rapid procedure which avoids most of problems of denaturing fragile molecules in chromatographic beads. ATPS provide a gentle environment for biologically active proteins and may be employed on a large scale (1, 2, 9).

In order to have a high yield, recovery and also a good purification factor for a target protein, a composition of the ATPS has to be selected. Compositions of desirable systems are those which promote high values for the partition coefficients (K), where the target protein is primarily extracted in one of the phases and the main contaminants are extracted in the other phase. The partition coefficient of a particle, K, is defined as the ratio of the concentration of the partitioned substance in the top phase to the concentration in the bottom phase. For extreme K values of the target protein, most of the contaminants should partition to the bottom (higher density) phase and for very low K values of the target protein, the majority of the contaminants would have to partition towards the top (lower density) phase for a good separation to occur. By following the above approach for the designing of compositions of the ATPS, it is possible to quantitatively extract the desirable protein from one of the phases with minimal concentration of contaminant molecules. A single-extraction step is usually sufficient for removal of noncritical contaminants, such as albumin in albumin-free pharmaceutical products. However, critical contaminants can be eliminated by countercurrent extraction, using a fresh pure phase from identical systems without the sample. They may also be removed by polishing the material collected from the enriched phase by using another process, usually a chromatographic step (2, 6, 7, 9).

The main factors which usually affect the partitioning of the target protein and its contaminants, are the molecular weight and the concentration of the polymers, the pH, the type of salt used for phase separation, e.g., phosphate, citrate or sulfate, and the addition of extra salt.

The composition of the required ATPS can be found by investigating the effects of a single parameter on the partition coefficients (one by one) or by applying an experimental factorial design.

Liquid-liquid partitioning of proteins in aqueous two-phase systems (ATPS) can be an efficient and versatile method for the separation and purification of proteins. The systems are usually composed of aqueous solutions of two polymers, polyethyleneglycol (PEG) and dextran or of PEG and a lyotropic salt, usually phosphate, citrate or sulfate (4, 6).

The advantages of two-phase partitioning, when compared with other purification processes, include rapid and continuous operation leading to high yields, ease of scale-up, low material costs and polymer and salt recycling (9), minimal protein denaturation and the possibility of having particulate material in the process.

This work was organized in two parts, the first investigated the partitioning of three different b-galactosidases in PEG/phosphate systems in a simple single-step procedure and aimed to achieve a high purification factor. The second part investigated the use of an affinity ATPS for the purification of the enzyme.

2 — MATERIAL AND METHODS

The b-galactosidases investigated were from three microorganisms: Eschericha coli, Klueveromyces lactis and Aspergillus orizae.

2.1 – Production of b-galactosidases

For Escherichia coli b-galactosidase production, E. coli was grown at 37°C for 24 h in the following culture medium: 20.0 (g/L) bacto tryptone, 5.0 (g/L) lactose, 4.5 (g/L) KH₂PO₄, 4.0 (g/L) K₂HPO₄ and 5.0 (g/L) NaCl with pH adjusted to 6.9. The fermented culture broth was centrifuged at 2,000 g for 20 min. The cells were broken with chloroform + phosphate buffer and magneticly stirred for an hour (twice) in order to release the intracellular target enzyme. The aqueous supernatant was used as the source of crude b-galactosidase. The enzyme from Klueveromyces lactis (Lactomax 50) was a gift from Solvay (Buenos Aires, Argentina) and the Aspergillus orizae enzyme was purchased from Sigma (St.Louis, USA). All the other reagents used were of an analytical grade.

2.2 – Aqueous two-phase systems preparation

The aqueous two-fases phosphate systems were prepared according to Franco et al. (6) from stock solutions of 40% polyethyleneglycol (w/w) of four different molecular weights (PEG 1,500, 4,000, 6,000 and 8,000), 40% phosphate (w/w) and 25% dextran T505,000 (w/w). Phosphate stock solutions were formulated with different ratios of K₂HPO₄ to KH₂PO₄ to give the desired pH values.

2.3 – Determination of b-galactosidase activity

The b-galactosidase activities of the three microorganisms were determined by using o-nytrophenyl-b-galactopyranoside (ONPG) as substrate and by reading the formed colored complex at 420 nm. The enzyme from E. coli was assayed at 37°C, the enzyme from K. lactis at 45°C and the one from A. orizae at 30°C. One b-galactosidase unit corresponds to 1.0 micromol of orthophenol released per minute under each given condition.

2.4 – Partitioning of b-galactosidase

One hundred µl of enzyme were mixed with the ATPS, using a Vortex for 1 minute. Phase separation was achieved by centrifugation for 3 min. at 3,000 g and the phases were carefully separated and the interface of each tube discharged. A known volume of each phase was transferred and the activity was determined. The partition coefficient (K) was calculated from the ratio of the b-galactosidaseactivity found in the top phase and the activity found in the bottom phase.

2.5 – BSA standard curve

The protein concentration was measured using the dye-binding technique of Sedmark and Grossberg (14). Fifty to 100 µl of each BSA solution were transferred (in triplicate) to a cuvette containing 2.4 ml of water and 1.0 ml of Coomassie blue solution and mixed well, and the OD₅₉₅ was read with a spectrophotometer, against a blank which has 50 µl of water instead of BSA. BSA concentrations were plotted against the OD₅₉₅.

2.6 – Partitioning of total proteins

Fifty to 100 µl of the top phase were transferred from each prepared system to a cuvette containing 2.4 ml of water and 1.0 ml of Coomassie blue solution and mixed well, and the OD₅₉₅ was read with a spectrophotometer, against a blank which has 50 µl of a top phase of a system which has been equally prepared without any sample, instead of BSA. The blanks were done to correct the interference of the phase components. The procedure was repeated for the bottom phase of each system. A BSA standard curve was used to calculate protein concentration. The partition coefficient, K, was calculated as the ratio of protein in the top phase to that in the bottom phase at room temperature.

2.7 – Synthesis of modified PEG

The modified PEG was synthesized according to Delgado et al. (5) and Nilson and Mosbach (10). The affinity ligand p-aminophenyl 1-thio-b-D-galactopyranoside (APGP) was bound to PEG 4000 in two steps. Firstly PEG 4000 was dried by azeotropic distillation in toluene and then activated with 2,2,2-trifluoroetanosulphonyl (tresyl chloride from Sigma) to form the precursor PEG-tresilated (TPEG). TPEG was washed twice with HCl-ethanol (250:1, v:v), precipitated at 2°C and kept in a dissecator. In the second reaction step, TPEG reacted with APGP in phosphate buffer (pH 7.5) for five hours. Samples were taken each hour for measuring the effect of the ligand binding upon the partition coefficient of the enzyme. The reaction was stopped with 0.18 g tris.

2.8 – Specific b-galactosidase activity (SA_b-gal)

It is defined as the ratio of enzyme activity (U/ml) to the total protein concentration (mg/ml) and is expressed in U/mg of protein (equation 1).

2.9 – Purification factor (PF)

ThePF concept has been used in this work as a measurement to follow the purification operations, according to several authors (8, 13), and is defined as the ratio of the specific b-galactosidase activity after a purification step to the initial specific b-galactosidase activity (from the aqueous enzyme extract, or from a previous purification step) (equation 2).

The recovery of the enzyme, R, is defined as the ratio of the enzyme activity collected into an aqueous phase after partitioning to the total enzyme activity added to the system (equation 3).

3 — RESULTS AND DISCUSSION

3.1 – Partitioning in PEG/phosphate systems

b-galactosidases of different origins, from E. coli, K. lactis and A. orizae, were partitioned in ATPS composed of PEG and phosphate. Several different parameters were selected for influencing the partitioning of proteins in ATPS, the most effective usually being the molecular weight (MW) of PEG, PEG and salt concentration, pH and ionic strength and the presence of a specific ligand in the system. The effect of PEG molecular weight (MW) upon the partition coefficient of the b-galactosidases was firstly investigated in this work (Table 1). In order to achieve a good separation of the b-galactosidase and its main contaminants, it would be desirable to find an ATPS where they are mostly extracted in opposite phases. The activity in the aqueous E. coli b-galactosidase extract was 630 U/ml and protein concentration was 20 mg/ml; therefore, the SA_b-gal was 31 U/mg of protein. The volume ratio of the phases was approximately 1.0 for all the systems. The volume of enzyme added to each ATPS was 100 µl (63 U). Therefore, PF was calculated as the ratio of the specific b-galactosidase activity in the aqueous phase (after a partitioning purification step) to the initial specific b-galactosidase activity.

Figure 1 represents the E. coli b-galactosidase partitioning in PEG/phosphate systems. It can be observed that the molecular weight of PEG has a significant effect upon the partitioning of E. coli b-galactosidase, showing that K_b-gal was highest in PEG 1,500 and PEG 6,000/phosphate. However, as K_prot was also highest in PEG 1,500/phosphate, the PF achieved was the lowest. The purification factors increased with PEG MW. PF were 2.5, 6.4, 8.0 and 9.7 in systems composed of PEG 1,500, 4,000, 6,000 and 8,000/phosphate, respectively. Very high recoveries were achieved for the enzyme in the top phase of the systems: 96%, 90%, 95% and 72% of b-galactosidase were collected in PEG 1,500, PEG 4,000, PEG 6,000 and PEG 8,000 top phases, respectively. As total protein partitioning is also affected by PEG molecular weight, remaining mostly at the bottom phosphate-rich phase. PEG 8,000/phosphate was the system with the highest PF, 9.7, despite its lowest enzyme partitioning coefficient (K=19.0). K. lactis and A. orizae b-galactosidases did not seem to partition towards the top phase, independently of the PEG MW. The PEG/phosphate systems investigated were not able to separate b-galactosidase from the main contaminants. The addition of sodium chloride to PEG/salt and to PEG/dextran systems is commonly utilized to improve protein extraction and separation. However, concentrations as high as 12% NaCl (w/w) were added to the systems without any improvement in the purification of K. lactis and A. orizae b-galactosidases. It seems that K. lactis b-galactosidase is the least hydrophobic enzyme of the three investigated, since according to Franco et al. (6,7) only very hydrophilic proteins do not have their partition coefficients raised by the addition of NaCl to PEG/phosphate systems. The very low K (0.01 to 0.05) of K. lactis b-galactosidase can also be considered an indication of low hydrophobicity.

3.2 – Affinity partitioning

In order to improve the extraction and separation of the K. lactis b-galactosidase, an affinity ATPS was developed. The system consisted of the APGP biospecific ligand chemically attached to PEG (two-step reaction) and dextran T505,000. In the first step PEG hydroxyls had to be activated with tresyl chloride to become more reactive. In the second step the ligand APGP was finally bound to PEG.

The elemental analysis for sulfur indicated that 74% of total hydroxyl groups in PEG had been transformed into tresyl esters in the first activation step. This activation rate is slightly lower than the rates achieved by Nilson and Mosbach (10) and by Delgado et al. (5), indicating that some of the tresyl chloride molecules might have been hydrolyzed by water before reacting with PEG hydroxyls despite laboratory precautions taken to achieve water-free solvents and conditions. Nilson and Mosbach (10) have activated 80% of PEG hydroxyl groups with tresyl chloride and Delgado et al. (5) have achieved approximately 83% activation.

The amount of APGP bound to PEG was determined indirectly by preparing an ATPS composed of the reacted polymer and dextran T500 (6% PEG4000 + 12% dextran) for the partitioning of b-galactosidase from Klueveromyces lactis. A direct method to control the chemical binding of APGP to TPEG using HPLC was previously investigated. It was not possible to detect the decrease of APGP concentration in the supernatant and the elution times of TPEG and APGP-PEG were found to be very close (gel permeation chromatography, Bio-Gel SEC 30XL column). The results from Table 2 show that the partition coefficient of b-galactosidase increased more than 250% for the highest ratio of APGP attached to TPEG. Approximately 49 units of b-galactosidase were recovered (83%) in the top phase of the system with the highest APGP content (polymer which reacted for 5 hours with APGP) from 60 units which had been previously added to the system. It was also found that more than 80% of the contaminant proteins had partitioned towards the bottom phase. Therefore, a 1.6-fold increase in the purification factor was achieved in just a single step of partitioning in this system.

Park et al. (11) have extracted and purified a b-galactosidase from Aspergillus orizae by using three different chromatographic steps, achieving very low yields despite a high specific activity. Pastore and Park (12) have purified a b-galactosidase from Scopulariopsis sp by precipitation with ammonium sulfate and two chromatographic steps leading to a 4% yield of the desired pure enzyme. Stred´ansky et al. (15) have optimized the enzyme extraction from Klueveromyces lactis by choosing the ideal pH and temperature values, as well as the concentrations of phosphate and chloroform, in order to release 95% of the intracellular b-galactosidase. Veide et al. (16) have developed an isolation and purification industrial process for E. coli b-galactosidase by partitioning in PEG4000/potassium phosphate systems, followed by an ultrafiltration step for the recycling of the salt-rich phase. They were able to recover 95% of the b-galactosidase. The only enzyme which was efficiently purified in simple ATPS was from E. coli, following the same trend as in the previous authors, probably due to its higher surface hydrophobicity, since the other two hydrophilic yeast b-galactosidases do not partition towards the top PEG-rich phase almost independently of the polymer molecular weight or the addition of extra salt.

It seems clear that partitioning in ATPS can be a very effective way of purifying and concentrating enzymes and other biomolecules. It can be highly improved by designing efficient and specific bioligands which should not be expensive and should need to be recycled (i.e. ultrafiltration). Research on affinity techniques, binding chemistry and the designing of specific equipments for extraction in ATPS will be of great help in the improvement of downstream processing of enzymes.

4 — CONCLUSIONS

A significative effect of the molecular weight of PEG upon the partitioning of E. coli b-galactosidase was observed. PEG 6,000 and PEG 8,000/phosphate systems gave better separations of the E. coli enzyme from the main contaminants and higher purification factor values. Recoveries of 95 and 72% of the enzyme and purification factors of 8.0 to 9.7 were achieved in systems composed of PEG 6,000/phosphate and PEG 8,000/phosphate, respectively, in a single-partitioning step.

However, K. lactis and A. orizae b-galactosidases were not purified in PEG/salt systems independently of the PEG MW, and an affinity ATPS was developed for their purification. It was possible to recover 83% of K. lactis b-galactosidase in the top APGP-PEG 4000 phase, with a 1.6-fold increase in the purification factor, by using an affinity ligand attached to PEG in the PEG/dextran T505,000 system. More than 80% of the contaminant proteins were collected in the dextran-rich bottom phase.

5 — REFERENCES

(3) BECERRA, M. AND SISO, M.I.G. Yeast b-galactosidase in solid-state fermentation. Enzyme Microb.Technol., 19:39-44, 1996.

(12) PASTORE, G.M. and PARK, Y.K. Purification and characterization of b-galactosidase from Scopulariopsis sp. J. Ferm. Technol., 58(1):79-81, 1980.

(15) STRED´ANSKY, M., TOMÁSKA, M., STURDIK, E. and KREMMINCKY, L. Optimization of b-galactosidase extraction from Kluyveromyces marxianus. Enz.Microb.Technol., 15:1063-1065, 1993.

(16) VEIDE, A., STRANDBERG, L. and ENFORS, S-O. Extraction of b-galactosidase fused protein A in ATPS. Enz.Microb.Technol., 9:730-738, 1987.

6 — ACKNOWLEDGMENT

The financial assistance received from CNPq and FAPESP in the form of scholarships for F.A.T.P., M.E.S. and C.P. and for support of the research project are gratefully acknowledged.

² To whom all correspondence should be addressed Chemical Engineering, State University of Campinas (Unicamp), PO Box 6066, Campinas 13081-970. Brazil. e-mail: [e-mail]

^* Towhom correspondence should be addressed.

• (1) ALBERTSSON. P.-Å. Partition of Cell Particles and Macromolecules 2^nd ed. Interscience, New York, 1971.
• (2) ASENJO, J.A. AND PATRICK, I. Large-scale Protein Purification, chap.1 In "Protein Purification Applications - A Practical Approach", E.L.V. Harris and S.Angal, eds. IRL Press, Oxford, UK. 1995.
• (4) CASCONE, O., ANDREWS, B.A. and ASENJO, J.A. Partitioning and purification of thaumatin in aqueous two-phase systems. Enzyme Microb.Technol, 13:629-635, 1991.
• (5) DELGADO, C., PATEL, J.N., FRANCIS, G.E. and FISHER, D. Coupling of poly(ethyleneglycol) to albumin under very mild conditions by activation with chloride: characterization of the conjugate by partitioning in aqueous two-phase systems. Biotech. Appl. Biochem, 12:110-128, 1990.
• (6) FRANCO, T.T., ANDREWS, A.T. and ASENJO, J.A. Use of chemically modified proteins to study the effect of a single protein characteristics in aqueous two-phase systems. Effect of surface hydrophobicity. Biotech.Bioeng, 49:300-308, 1996.
• (7) FRANCO, T.T., ANDREWS, A.T. and ASENJO, J.A. Use of chemically modified proteins to study the effect of a single protein characteristics in aqueous two-phase systems. Effect of surface charge. Biotech.Bioeng, 49:309-315, 1996.
• (8) FROUND, S.J. Example Purifications. In "Protein Purification Applications - A Practical Approach",. E.L.V. Harris and S.Angal, eds. IRL Press, Oxford, UK. 1995.
• (9) KULA, M-R. Trends and future of aqueous two-phase extraction. Bioseparation, 1:181-189, 1990.
• (10) NILSSON, K. and MOSBACH, K. Immobilization of ligands with organic sulphonyl chlorides. Methods in Enzymology, 104:56-69, 1984.
• (11)PARK, Y.K., DE SANTI, M.S.S., PASTORE, G.M. Production and characterization of ß-galactosidase from Aspergillus oryzae. Journal of Food Science, 44(1):100-103, 1979.  
• (13)PRICE, N.C. and STEVENS, L. Fundamentals of Enzymology 2^nd ed. Oxford Science Publications. 1989.
• (14) SEDMAK, J. J., GROSSBERG, S.E. A rapid, sensitive, and versatile assay for protein using coomassie brilliant blue G250. Analytical Biochemistry, 79(1): 544-552, 1977.

¹

Recebido para publicação em 10/07/97. Aceito para publicação em 07/10/97.

Datas de Publicação

• Publicação nesta coleção
  14 Dez 2004
• Data do Fascículo
  Dez 1997

Histórico

• Recebido
  10 Jul 1997
• Aceito
  07 Out 1997