Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
1998-01-08
2003-10-28
Naff, David M. (Department: 1651)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S041000, C435S183000, C530S422000
Reexamination Certificate
active
06638749
ABSTRACT:
BACKGROUND OF THE INVENTION
As is well known, purification of proteins from cell broth is usually a multi-step process involving cell separation, concentration and fractionation by methods such as chromatography. From a process standpoint, particularly with regard to throughput, it would be advantageous to directly extract proteins from whole broth by means such as micellar liquid-liquid extraction, however, lack of selectivity and product recovery difficulties have until now precluded the use of such purification methods on a large scale. As described by Hatton, T. A. et al. in
Surfactant Based Separation Processes
, Scamehorn, J. F. and Harwell, J. H., eds., p. 5 (1989), surfactants can be employed to extract proteins from a buffer to an organic phase, generally through manipulation of the physiochemical properties of the aqueous phase such as pH, ionic strength, etc. So far the organics which have been found to be useful are hydrocarbons, which are toxic and flammable, and thus undesirable from a commercial perspective.
Reverse micelles have also been used to solubilize proteins of industrial relevance. In reverse micelles, or water-in-oil microemulsions, polar molecules such as water, salts and proteins can be solubilized by the polar groups of organic soluble surfactants. Aires-Barros, M. R. et al.,
Biotech. Bioeng
. 38:1302 (1991), describes the separation and purification of two lipases using liquid-liquid extraction which was carried out in isooctane and utilized sodium di-2-ethylhexylsulfosuccinate as the surfactant. Dekker, M. et al.,
Chem. Eng. J
. 33B:27 (1986), describe the recovery of &agr;-amylase by liquid-liquid extraction into isooctane using trioctylmethylammonium chloride as the surfactant. Further, surfactant systems have been used to extract proteins from complex matrices, including fermentation broth and dried solids. (See Rahaman, R. S. et al.,
Biotech. Progr
. 4:218 (1988); and Leser, M. E. et al.,
Chimia
44:270 (1990), respectively.)
It has been demonstrated that micellar systems allow not only extraction of proteins from aqueous solution, but also separation of certain protein mixtures. Goklen, K. E. et al.,
Sep. Sci. Tech
. 22:831 (1987), show that separation of protein mixtures has been achieved through control of pH. Thus, proteins are separated by adjusting the pH of the micellar phase relative to the protein's isoelectric point(s). Salt concentration (which regulates water content, and thus micelle size) and the use of amphiphilic compounds which incorporate affinity ligands are another way to control separation. Work by Hatton et al. (supra) has shown that the affinity ligand approach yields a stronger interaction between the extracted protein and the micellar phase, and thus, allows protein solubilization over a wider range of pH and salt concentration.
While intriguing, micellar extraction of proteins cannot presently compete with other separation technologies owing to low overall protein recovery and difficulty in re-use of the organic phase, which is itself tied to the mechanism by which the protein is back-extracted. John, V. J. et al.,
J. Supercrit
. Fl. 4:238 (1991), describe an approach to circumvent the back-extraction problem through the addition of a highly compressible organic component to the microemulsion. In this approach the formation of clathrate hydrates caused by the added organic removed water from the micelles and precipitated the protein. This technique, while successful on a small scale, does not entirely resolve the problem of protein recovery given that a let-down of the pressure permits resolubilization of both water and protein. Molecular sieves have been employed in conventional liquid emulsions for similar purposes. A final approach to addressing the back-extraction problem relates to increasing the salt concentration, or adding disrupting agents such as ethyl acetate or ethanol to dewater the reverse micelles, thus inducing protein precipitation from the emulsion. Such tactics may damage certain proteins, and none of these strategies address the overall problem of use of large volumes of an organic, solvent in contact with an aqueous phase. Clearly, the organic will contaminate the aqueous phase to some extent, which will require remediation prior to discharge.
One should be able to induce protein migration by varying the organic phase properties, as well as those of the aqueous phase. (See Hatton, T. A. et al., supra, for discussion of variables which induce protein migration.) As first shown by Smith, R. D. et al.,
J. Phys. Chem
. 95:3253 (1991), variation of the pressure in a near- or supercritical fluid propane-based emulsion will indeed prompt proteins to migrate from the aqueous phase to the organic phase. Further, solubilization of a protein without a significant loss in activity in a propane-based emulsion allows extraction and recovery through pressure manipulation. (Ayala et al. (1992)
Biotechnology and Bioengineering
39:806-814; Ayala et al. (1992)
Biotechnology
10:1584-1588.)
Despite these favorable results, use of propane on a large scale is precluded by safety and environmental considerations. Indeed, use of most any organic solvent on a large scale could be unacceptable or very costly, owing to regulations regarding volatile organic chemical (VOC) emulsions, plus toxicity and safety issues.
In contrast to conventional organic solvents, carbon dioxide is inexpensive, occurs naturally in large quantities, is relatively non-toxic, is non-flammable and is not considered to be a VOC by federal authorities. In addition, carbon dioxide has a low critical temperature (~31° C.), allowing it to be used for extraction of thermally labile material. Consequently, carbon dioxide has been identified as an environmentally-benign organic solvent in such diverse areas as analytical chromatography, biocatalysis, polymerization and extraction of thermally-labile constituents from natural products. Because carbon dioxide is a gas under ambient conditions, reduction of the pressure to atmospheric in carbon dioxide-based solutions induces complete precipitation of solute, rendering solute concentration/recovery and solvent recycle operations somewhat easier than in conventional liquid systems. However, despite its inherent physical property advantages, carbon dioxide by itself is a relatively non-polar material, and thus will not solubilize highly polar and hydrophilic solutes.
In the late 1980's researchers at the University of Texas-Austin and Battelle's Northwest Laboratories investigated the use of surfactants to improve the solubility of polar solutes in non-polar supercritical fluids. Formation of reverse micelles in supercritical alkanes did indeed dramatically increase the supercritical fluid's ability to solubilize amino acids, water-soluble polymers, proteins and metal-containing compounds. Gale, R. W.; Fulton, J. L.; Smith, R. D. (1987)
J. Am. Chem. Soc
. 109:920 and Hoefeling, T. A. et al. (1991)
J. Phys. Chem
. 95:7127. However, extension of the use of surfactants to environmentally-benign carbon dioxide was blocked by the experimental observation that commercially available ionic amphiphiles, while highly soluble in alkanes such as ethane and propane, exhibit poor to negligible solubility in carbon dioxide at moderate pressures (10-500 bar). These findings were discussed in Consani, K. A.,
J. Supercrit
. Fl. 3:51 (1990). Thus, in carbon dioxide/water mixtures, these conventional hydrocarbon surfactants tend to partition to the aqueous phase, forming normal micelles. Nonionic ethoxylated alcohols as well as other surfactants, while somewhat soluble in carbon dioxide, exhibit poor water absorption capability, making these unacceptable as surfactants to use with carbon dioxide for solubilizing biomolecules such as proteins. The results of the work described in this reference are summarized in Table 1 in the Experimental section herein.
To address the problem of the insolubility of standard surfactants in carbon dioxide the applicants have investigated the rational design and
Becker Nathaniel T.
Beckman Eric J.
Ghenciu Eliador J.
Russell Alan J.
Steele Landon M.
Genencor International Inc.
Genencor International Inc.
Naff David M.
University of Pittsburgh
Ware Deborah K.
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