Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for...
Patent
1994-12-08
1998-03-31
Lankford, Jr., Leon B.
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
435188, 435814, 435816, 210696, C12N 900
Patent
active
057337640
DESCRIPTION:
BRIEF SUMMARY
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a 371 of PCT/DK93/00229 filed Jul. 6, 1993, published as WO94/01537 Jan. 20, 1994 which is incorporated herein by reference.
In order to purify or concentrate an aqueous enzyme solution, especially an enzymatic fermentation broth, different purification methods are known in the art. Examples are salting-out and treatment with activated carbon. However, the selectivity, with which these purification methods separate enzymes from carbohydrates, non enzymatic proteins, colored compounds, and other impurities is open to improvement.
Thus, the purpose of the invention is the provision of a method for purification of an aqueous enzyme solution by means of which the enzymes can be effectively separated from carbohydrates, non enzymatic proteins, colored compounds, and other impurities. Usual sources of aqueous enzyme solutions are fermentation broths or filtered fermentation broths.
The method for purification of an aqueous enzyme solution according to the invention is characterized by the fact that a soluble aluminate is added to the aqueous enzyme solution, and that simultaneously an acid is added in such amounts that the pH-value of the solution is maintained between 4 and 10 and that the pH-value differs at least 1 pH unit from the isoelectric point of the enzyme to be purified, whereby the soluble aluminate is added to the solution in an amount between a maximum amount, which will precipitate no more than a negligible amount of enzyme and a minimal amount, which will leave no more than a negligible amount of impurities, mainly carbohydrates, in solution, and that the supernatant subsequently is separated from the precipitate, which is discarded.
It has been found that the precipitation process proceeds in the most efficient manner at lower pH-values. However, most enzymes exhibit low stability at low pH-values. Thus, a compromise pH-value with a reasonably efficient process and a reasonably good enzyme stability has to be chosen.
According to the invention it surprisingly has been found that the "window", i.e. the interval of added aluminate, in which practically no enzymes are precipitated and practically all impurities are precipitated, is larger than for other precipitating agents, if the precipitation is carried out by means of the method according to the invention.
It is to be understood that the addition of the soluble aluminate to the pH-adjusted enzyme solution with subsequent precipitation of aluminum hydroxide will change the pH, but that according to the invention the pH-value will be readjusted with formic acid, if the changed pH-value should not fulfil the above indicated conditions, i.e. if the changed pH-value should not lie in the interval between 4 and 10, and if it should not differ at least 1 pH unit from the isoelectric point of the enzyme to be purified.
In this specification with claims "a soluble aluminate" means an aluminate, the solubility in aqueous medium of which is above 100 g/l of solution at a temperature of 25.degree. C. An example is sodium aluminate.
The maximum amount of aluminate, which will precipitate no more than a negligible amount of enzyme is dependent upon the kind and the concentration of the enzyme, and it will normally be around 5% w/w aluminate in regard to the solution.
The minimal amount of aluminate, which will precipitate no more than a negligible amount of impurities, mainly carbohydrates, is dependent upon the kind and concentration of the impurities, and it will normally be around 0.5% w/w in regard to the solution.
A few enzymes will tend to precipitate to a certain degree already with addition of the soluble aluminate in an amount of 0.8% aluminate in regard to the solution, and such enzymes are not very well suited for this invention.
U.S. Pat. No. 3,795,586 describes a purification method for enzymes, in which aluminum hydroxide and other insoluble compounds are precipiated in situ. However, in all examples, in which aluminum hydroxide is formed, aluminum sulphate was used, and it has been f
REFERENCES:
patent: 3664926 (1972-05-01), Grabner et al.
patent: 3795586 (1974-03-01), Ziffer
patent: 4237231 (1980-12-01), Jackson et al.
patent: 4711739 (1987-12-01), Kandathil
patent: 4994200 (1991-02-01), Disch et al.
patent: 5385837 (1995-01-01), Boyer et al.
Gregg, Esq. Valeta
Lankford , Jr. Leon B.
Novo Nordisk A S
Zelson Esq. Steve T.
LandOfFree
Method for purification of an aqueous enzyme solution does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Method for purification of an aqueous enzyme solution, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method for purification of an aqueous enzyme solution will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-51102