Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for...
Patent
1995-09-07
1998-02-17
Lankford, Jr., Leon B.
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
435198, 435814, 435816, C12N 900, C12N 920
Patent
active
057190488
DESCRIPTION:
BRIEF SUMMARY
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a 371 of PCT/DK94/00132 filed Mar. 29, 1994, which is incorporated herein by reference.
TECHNICAL FIELD
The present invention relates to a method of separating an enzyme from an aqueous solution comprising this enzyme in mixture with other proteins, and recovery of the desired enzyme on crystalline form.
BACKGROUND ART
Enzymes are usually provided as liquids or amorphous materials for industrial purposes. When not provided as liquids, they are usually provided as amorphous materials, because the known methods for crystallization of enzymes are usually regarded as too expensive to be used in an industrial scale. This is due to the assumption that crystals can only be grown from very pure and concentrated solutions. In fact, capability of forming crystals is by protein chemists often regarded as evidence of high purity of a protein solution.
Due to the high purity of enzyme crystals, the provision of a cheap and simple method for crystallization of enzymes, which is easily adaptable to an industrial scale, is clearly a desideratum in the industry.
There is an abundance of literature concerning crystallization of enzymes. Characteristic features of the hitherto known crystallization processes are relatively pure and concentrated initial solutions, low yield, very long crystallization time, high consumption of chemicals including organic solvents, and poor industrial adaptability.
The use of low conductivity and pH around pI is in itself far from a new method for crystallization of proteins. It is a technique well described in the literature and is often used for preparation of single crystals for X-ray diffraction from highly purified protein solutions. A. McPherson describes in his book "Preparation and Analysis of Protein Crystals" (Wiley, New York, 1982) how this technique, also known as "salting in" can be used in crystallization. Since then several others have used this method, but only for crystallization from purified solutions. In fact, it is often reported that impurities, and especially other proteins, are regarded as highly undesirable (for reference see A. McPherson in Eur. J. Biochem. 289, 1990, pp. 1-23). Thus, the technique in itself is not new, but nobody has ever used it as a purification technique for purification of enzymes in mixtures with other proteins. As such the surprisingly good adaptability on very impure solutions is what makes this technique economically feasible, in that it does not require prepurification by e.g. chromatographic methods.
It is important to distinguish between crystallization and precipitation, the latter resulting in the less pure amorphous form.
In EP patent 193,046 a method for the recovery of an enzyme from a solution thereof is disclosed. By this method the enzyme containing solution is concentrated to supersaturation at a pH value in a range near the isoelectric point of the enzyme, and crystallization is induced. The presence of salt or organic solvents has been thought essential to any crystallization method. In the above captioned EP 193,046 it is stated that the introduction of salts or organic solvents is unnecessary for the method described, but from the experiments described it is also evident that the enzymes investigated in this patent publication crystallize best in the presence of high salt concentrations, i.e. when the ultrafiltration is followed by vacuum evaporation. Moreover, the method is adapted only to amylases, long crystallization times are required, and the method leads to undesirable amorphous and micro crystalline precipitations.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide a process for the recovery of crystalline enzymes, which process does not require the addition of salts or organic solvents, which permits short crystallization times and high yields, and which is simple and cheap, and compatible to industrial requirements.
Accordingly, the present invention provides a method of separating an enzyme from an aqueous solution compris
REFERENCES:
patent: 3642582 (1972-02-01), McClary
Laustsen Mads Aage
Nilsson Birgitte Mahler
Pahle Christine
Gregg, Esq. Valeta
Lankford , Jr. Leon B.
Novo Nordisk A S
Zelson Esq. Steve T.
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