Engineering and Manufacturing for Biotechnology - Marcel Hofman & Philippe Thonart

Silvia Andrea Camperi, _ Mariano Grasselli and Osvaldo Cascone

3. Results and discussion

3.1. CHROMATOGRAPHIC CHARACTERISATION OF THE DERIVATISED MEMBRANES

IDA was immobilised on epoxy-activated PSU membranes. The Cu(II)IDA-membranes had a copper saturation capacity of 60 and a pure water SV of 234 at a filtration pressure of 1 bar.

Non-selective adsorption of biomolecules on the derivatised hollow fibres was assessed through histidine and lysozyme adsorption onto the IDA-membranes in the absence of copper. A negligible biomolecule adsorption was observed.

The adsorption isotherms for histidine, lysozyme, myoglobin and haemoglobin binding to Cu(II)IDA membranes showed a good fit of experimental data to a Langmuir-type

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isotherm as is indicated in Figure 3. Table 1 shows the calculated maximum adsorption capacity (qm) and the values for the dissociation constant (Kd) for the pure adsorbates.

The molar ratio of histidine adsorbed to Cu(II)-IDA was approximately 0.5 thus indicating that only 50% of the immobilised ligand was accessible to histidine. This reduced capacity was attributed to inaccessibility of the chelated copper ions and steric

ml of Chelating Sepharose Fast Flow and TSK Gel Chelate respectively).

The accessibility of the ligand immobilised onto the membrane for interaction with proteins decreased with the rise in molecular weight of the protein - a result of the molecular-sifting properties of the membrane. On the other hand, the adsorption of proteins to IDA membranes could block the access to other copper sites thus explaining why the IDA to protein molar ratio increased with the protein molecular weight (Belew, etal., 1987).

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Table 1 shows that the Kd values decrease with the number of surface histidines. Many chromatographic studies on protein and peptides demonstrated that retention in Me(II)-

IDA adsorbents is dictated primarily by the availability of histidyl residues (Arnold, 1991).

The adsorption isotherm of the PE binding showed a qm of 8000 U/ml and a Kd value of 20.3 U/ml.

3.2. PROPERTIES OF THE HOLLOW-FIBRE MEMBRANE MODULE

The SV for the module was the same as that of a single hollow fibre. This result indicates an advantage of membrane chromatography over conventional bead-packed columns: scale-up of the former does not require a high-pressure pump whereas scaleup of the latter does so, unless a thin column with a large diameter, i.e., the equivalent of a functional porous membrane, is used.

The chemical stability of the IDA-membrane cartridge was examined by repeated adsorption and elution cycles of lysozyme. The capacity of the module to adsorbed lysozyme stayed constant thus evidencing chemical stability of the ligand.

3.3. BREAKTHROUGH CURVES FOR PE AND PL ADSORPTION

Figure 4 shows the breakthrough curve for the adsorption of PE and PL working at a SV of 5 and with an input stream adsorbate concentration of 600.3 U/ml of PE and 289 U/ml of PL. The dynamic capacity of the column under these conditions was 7500 PE U/ml. PL was not adsorbed by the chromatographic membrane.

3.4. UTILISATION OF THE IDA-CARTRIDGE FOR PECTIC ENZYME

FRACTIONATION

In order to test the usefulness of the cartridge for pectic enzyme fractionation, the hollow-fibre cartridge (0.408 ml) was loaded with 3000 U of PE and 1445 U of PL (5 ml of Biocon Bioconcentrated Plus 23 mg/ml) at a SV of 5

Figure 5 shows the pattern obtained. 99 per cent of the PE activity was retained by the chromatographic matrix and eluted quantitatively with 0.1 M EDTA, pH 7.0, thus indicating that the fractionation procedure can be successfully scaled-up. The time of the fractionation process, 10 min was far shorter than when working with chelating soft gel: 50 min (Camperi et al., 1996) where lower flow rates must be used to allow mass transfer. The better hydrodynamic properties of the membranes resulted in an enormous saving 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).

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4. Conclusions

The high capacity of the membrane cartridge for PE and its excellent hydrodynamic properties allows a very fast fractionation of a commercial pectic enzyme preparation at a low operating pressure. The fraction passing through - containing all the PL activity loaded onto the cartridge - can be used directly to clarify fruit juice without production of methanol.

Acknowledgements

This work was supported by grants from the Universidad de Buenos Aires, the Consejo Nacional de Investigaciones Cientificas y Técnicas de la República Argentina (CONICET) and the Agencia Nacional de Promoción Cientifica y Tecnológica.

M.G. and O.C. are career researchers of the CONICET.

References

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Albersheim, P. (1966) Pectinlyase from fungi, in: Neufeld, E.F. and Ginsburg, V. (eds.), Methods in Enzymology, Academic Press, New York, vol. 8, pp.628-631.

Arnold, F.H. (1991) Metal-affinity separations: a new dimension in protein processing, Bio/Technology 9, 151-155

Belew, M., Yip, T.T., Anderson, L. and Porath, J. (1987) Interaction of proteins with immobilised

Quantitation and equilibrium constants by frontal analysis, J. Chromatog. 403, 197-206.

Brandt, S., Goffe, R.A., Kessler, S.B., O’Connor, J.L. and Zale, S.E. (1988) Membrane-based affinity technology for commercial scale purification, Bio/Technology 6, 779-782.

Camperi, S.A, Auday, R.B, Navarro del Cañizo, A.A.and Cascone, O. (1996) Study of variables involved in fungal pectic enzyme fractionation by immobilised metal ion affinity chromatography, Process

Biochem.31, 81-87.

Camperi, S.A., Navarro del Cañizo, A.A., Wolman, F.J., Smolko, E.E., Cascone, O. and Grasselli, M. (1999)

Protein adsorption onto tentacle cation-exchange hollow-fiber membranes, Biotechnol. Prog. 15, 500505.

Chase, H.A. (1984) Prediction of the performance of preparative affinity chromatography, J. Chromatog. 297, 179-202.

Hemdan, E.S., Zhao, Y., Sulkowski, E. and Porath, J. (1989) Surface topography of histidine residues: a facile probe by immobilised metal ion affinity chromatography, Proc Nat. Acad. Sci. USA 86, 18111815.

Kroner, K.H, Krause, S. and Deckwer, W.D. (1992) Cross-flow anwendung von affinitätsmembranen zur primärseparation von proteinen, Bioforum 12, 455-458.

Kubota, N.. Konno, Y., Saito, K., Sugita, K., Watanabe, K., Sugo, T. (1997) Comparison of protein adsorption onto porous hollow-fiber membrane and gel bead-packed bed, J. Chromatog. 782, 159-165.

Mueller-Schulte, D. and Daschek, W. (1995) Application of radiation grafted media for lectin affinity separation and urease immobilisation: a novel approach to tumour therapy and renal disease diagnosis,

Radiat. Phys Chem. 46, 1043-1047.

Navarro del Cañizo, A.A., Hours, R.A., Miranda, M.V. and Cascone, O. (1994) Fractionation of fungal pectic enzymes by immobilised metal ion affinity chromatography, J. Sci. FoodAgric. 64, 527-531.

Porath, J., Carlsson, J., Olsson, I. and Belfrage, G. (1975) Metal chelate affinity chromatography, a new approach to protein fractionation, Nature 258, 598-599.

Rombouts, F.M. and Pilnik, W. (1980) Pectic Enzymes, in: Rose AH (ed.) Economic Microbiology, Academic Press, London, vol. 5, pp. 228-282.

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Roper, D.K. and Lightfoot, E.N. (1995) Separation of biomolecules using adsorptive membranes. J.

Chromatog. A., 702, 3-26.

Saito, K., Ito, M, Yamagishi, H., Furusaki, S., Sugo, T. and Okamoto, J. (1989) Novel hollow fiber membrane for removal of metal ion during permeation: preparation by radiation-induced co-grafting of a cross-linked agent with reactive monomer, Ind. Eng. Chem. Res. 28, 1808-1812.

Szajer, I. and Szajer, Cz.(1982) Clarification of apple juices by pectin lyase from Penicillium paxilli,

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Vilariño, C., Del Giorgio, J.F., Hours, R.A. and Cascone, O. (1993) Spectrophotometric method for fungal pectin-esterase activity determination, Lebensm Wiss u Technol. 26, 107-110.

Wuenschell, G. E., Wen, E., Todd, R., Shhek, D. and Arnold, F. H. (1991) Aqueous two-phase metal affinity extraction of heam proteins. J. Chromatog. 543, 345-354.

Yamagishi, H., Saito, K., Furusaki, S., Sugo, T. and Ishigaki, Y. (1991) Introduction of high-density chelating group into a porous membrane without lowering the flux. Ind. Eng. Chem. Res. 30, 2235-

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ECONOMIC BENEFITS OF THE APPLICATION OF BIOTECHNOLOGY - EXAMPLES

MARLENE ETSCHMANN, PETER GEBHART AND DIETER SELL

DECHEMA e. V., Theodor-Heuss-Allee 25, D-60486 Frankfurt am Main,

Germany, fax: ++ 49 697564388, e-mail: sell@dechema.de

Summary

Biotechnological processes or process steps can substitute traditionally applied techniques. Of all the arguments to be considered when choosing a biotechnical process, improving the process economy is the most important one.

Different areas of application for biotechnology have been examined and different process alternatives (biotechnological vs. conventional processes) compared from an economic point of view.

Overview

Biotechnological methods are increasingly being used to substitute chemical processes in a wide range of industries. This even affects sectors where at first sight it may seem surprising to find biotechnology at work, for example in textile finishing or in pulp and paper production. Biotechnological methods may have innumerable advantages if looked at from a researcher’s point of view, but to be applied in practice they have to meet one stipulation: they have to be cheaper than the conventional process.

Environmental advantages are often only regarded as a pleasing side effect. A change in thinking still has to take place to the effect that costs for environmental protection should be considered during process design. Production integrated measures can reduce environmental costs dramatically compared to conventional end-of-pipe techniques, which are added to existing processes.

In the following, five examples are presented which demonstrate that it is economically and ecologically advantageous to replace a traditional process by one with one or more biotechnological steps. The first two, the production of 7-aminocephalosporanic acid and stone washing of jeans, are well known and were assessed some time ago. The next two, production of riboflavin and biopulping are

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© 2001 Kluwer Academic Publishers. Printed in the Netherlands.

Marlene Etschmann, Peter Gebhart and Dieter Sell

currently being implemented on an industrial scale. The third, bleach cleanup in textile finishing, has been applied for several years, but was investigated and calculated only recently by the authors.

1. Production of 7-aminocephalosporanic acid

7-aminocephalosporanic acid (7-ACA) is a pharmaceutical chemical, a key product for most semisynthetic cephalosporin antibiotics. Hoechst Marion Roussell, which uses it to produce several antibiotics, has developed a process based on biochemical catalysts. In the two-stage enzymatic synthesis D-alpha-amino acid oxidase and glutaryl amidase are used to form 7-ACA. The reactions are carried out at room temperature in an aqueous solution. In contrast to the chemical process no chlorinated hydrocarbons, toxic auxiliaries or heavy-metal salts are needed.

This is considerably more environmentally friendly than the chemical process formerly applied and reduces the percentage of process costs used for environmental protection (including waste incineration, purification of waste water and waste gas) from 21% to 1%. The absolute environmental protection costs are thus reduced by 90% per tonne of 7-ACA. (Wiesner et al. 1995, OECD 1998).

2. Stonewashing of jeans

Many of the 70 million jeans sold in Europe every year are stonewashed. This means that they are subjected to a washing process which locally abrades the indigo dyestuff from the cotton yarn and thus produces the desired look. In the past the abrasion effect on the garments originated from pumice stone. Nowadays there is an increasing tendency to use cellulase enzymes or a combination of pumice and enzymes. The

Institute for Applied Environmental Economics, The Hague, performed a life cycle assessment (LCA) to compare these three methods with respect to their environmental economic costs. The results show that enzymatic stone washing, also known as "biostoning", has numerous advantages, most of which are based on the simple fact that no stones are involved. Thus, there is

•no need to remove pumice fragments from the garments

•no need to landfill pumice sludge

•less machine damage

•lower maintenance costs

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to name just a few of the benefits.

Bio-stoning scores best in the comparison of environmental economic costs, however European textile finishers prefer to stone wash with a mix of enzymes and pumice as only the stones create the effect desired by the European consumer (Kothuis and Schelleman 1996).

3. Production of riboflavin

used as a vitamin in feed, food and pharmaceutical applications. Because of its yellow colour, it can also be used as a food colorant.

For large-scale applications, riboflavin is produced by a combination of chemical and fermentation processes. First, ribose is obtained by fermentation, then it is converted into riboflavin by a multistep chemical process. This procedure has been continuously improved in the last decades, with the principle still dating back to the 1930s. Only in the 1990s did a fundamental change take place, insofar as bacterial strains were developed which directly transform glucose to riboflavin. These strains were produced by a combination of classical mutation-selection and molecular biology methods. The quality of the riboflavin produced is equivalent or even slightly superior to riboflavin from chemical synthesis. The difference in the processes lies in the environmental impact: Biotechnological production uses almost exclusively renewable raw materials. The use of organic solvents and other chemical substances can be reduced, air emissions and waste are decreased by 36%. 25% less energy is used compared to the conventional process. These figures were determined by F. HoffmannLa Roche in the preliminary stages of the building of a commercial-scale riboflavin fermentation plant at Grenzach, Germany. Verification of the figures is anxiously awaited when the facility comes on stream in mid-2000 (Loon van, et. al. 1996,

Eggersdorfer et al. 1996, Bretzel et al. 1999).

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