Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
2000-01-24
2001-11-13
Saucier, Sandra E. (Department: 1681)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S200000, C435S201000, C435S219000
Reexamination Certificate
active
06316240
ABSTRACT:
FIELD OF INVENTION
The present invention relates to a method for recovering a glycosidase or a peptidase from a culture broth, from an insoluble state within said broth to a soluble state within said broth.
BACKGROUND ART
Recovery of a protein of interest from a culture broth may be hampered by the fact that a significant amount of the protein of interest is not in a solubilized form.
Said problem may occur when e.g. the protein of interest is bound to components in the sludge as such or due to the fact that a significant amount of the protein of interest is precipitated or crystallized prior to harvest from the culturebroth.
Sludge binding of a protein of interest denotes that the protein is bound to solids in the culture medium, such as cell solids or other solid components in the medium.
In relation to an effective recovery of the protein this may be a significant problem.
A number of methods have been applied for solving or minimizing said problem.
In many cases low or varying recovery yields have been accepted, in other cases the problem has been diminished through additions of e.g. ionic
onionic surfactants, salts, anti/defoaming agents, alcohols, substrate or substrate analogs for the enzyme in question.
In many cases such techniques have been of low efficiency, in other cases complicated procedures have been developed (e.g. liquid-liquid separation systems) requiring large amounts of agent(s) added.
Some examples are found in the recovery of lipases, which are enzymes with a high tendency towards binding to fermentation solids and/or potential filter aids used in recovery.
WO 97/23604 describes a method for recovering of a lipase from a fermentation broth.
Essential steps in the described method comprise the use of both a nonionic surfactant and an alcohol so that a final composition is obtained which contains the microbial protein (preferably a lipase), the nonionic surfactant and the alcohol. See e.g. claim 1 of said WO 97/23604 document.
EP 0574050A1 describes a method for recovering of a hydrophobic product, preferably a lipolytic enzyme, (see column 3, line 47-50) from a fermentation broth. Essential steps in the described method comprise the use of both a nonionic surfactant and a salt in the process. See e.g. claim 1.
Journal of Biotechnology,
26 (1992) 111-142 is a review article describing the state of the art for different purification strategies of a lipase. On page 129 it is mentioned that lipase purification is generally hampered by the problem of solubilization of the lipase. The article summarizes the state of the art solutions to this problem which comprise very complex purification strategies, such as Liquid-liquid extraction, aqueous two-phase systems, specific membrane processes, and immunopurification.
For proteins precipitated or crystallized prior to harvest of broth, no real efficient methods exist today. Examples of solubilizing with large amounts of solubilization agents are typically tried (e.g. urea). Other examples are solid-solid separation techniques wherein the precipitated or crystallized protein in question is separated from the other solids in the broth,—such separations are often not possible and are always complicated.
SUMMARY OF THE INVENTION
The problem to be solved by the present invention is to provide a simple and effective method for recovering a protein of interest, preferably a glycosidase or a peptidase, from a culture broth, wherein a significant amount of the protein of interest is NOT in a soluble form within said culture broth.
The invention is based on the present inventors having identified that a protein of interest in a culture broth, particularly a glycosidase or peptidase, wherein a significant amount of the protein is NOT in a soluble form at pH 7.5 within said culture broth, can very efficiently be recovered by the use of extreme pH values, i.e. pH values from pH 9.5 to pH 13.
Accordingly, the invention relates to a method for recovering a protein of interest, preferably a glycosidase or a peptidase, from a culture broth comprising a cell capable of producing the protein, wherein less than 80% of the protein of interest is in a soluble form at pH 7.5 within said culture solution, comprising:
adjusting the pH of the culture broth to a pH value from pH 9.5 to pH 13; and
b) removing the cells from the broth to obtain a solution comprising the protein of interest.
The term “a culture broth” denotes herein a culture medium comprising a protein of interest and a cell capable of producing the protein of interest. Said culture broth may comprise any further components, in particular components generally used for fermentation of a cell.
The term “wherein less than 80% of the protein of interest, at pH 7.5, is in a soluble form within said culture broth” is a term directed to the general problem to be solved herein, i.e. to recover a protein of interest wherein a significant amount of the protein of interest is not in solution.
Whether or not a “culture broth” complies with this criterion is herein defined by the fact that the concentration of the protein of interest in the supernatant of the culture solution has a concentration of less than 80% of the total concentration of the protein of interest in the culture solution as such. A supernatant is the aqueous medium obtained by, e.g., centrifugation under conditions sufficient to remove the cells, i.e., obtain a cell-containing pellet.
The term concentration denotes amount of protein (dry matter) per liter (e.g. g/l) or protein activity per liter.
The term “at pH 7.5” denotes herein that the supernatant is separated (preferably by centrifugation) from the culture solution as such at pH 7.5.
The concentration of the protein of interest in, respectively, the supernatant and the culture broth as such may then afterwards be measured at this pH 7.5 or at a different pH.
This will depend on the specific protein of interest and the specific culture broth and it is within the skilled persons general knowledge to decide this (see below for a further discussion of this).
Alternatively this may be expressed as:
A “culture solution” complies with the criteria set herein if:
Prot.
conc, sup.
/Prot.
conc. Cul
×100<80; at pH 7.5; wherein
Prot.
conc, sup.
is the concentration of the protein of interest in the supernatant of the culture broth;
Prot.
conc. Cul
is the total concentration of the protein of interest in the culture broth as such.
Preferably, said concentration of the protein of interest in the supernatant is less than 70%; more preferably less than 55%, even more preferably less than 40% and most preferably less than 25% of the concentration of the protein in the whole culture.
The actual methods for measuring the concentration of the protein of interest are done according to standard methods known in the art and adjusted to the specific protein of interest.
When the concentration of the enzyme is measured in the culture broth as such, the methods to be used shall of course be methods that solubilize the insoluble fraction of the protein of interest before the actual measurement of the concentration of the protein of interest within said culture broth.
Numerous such methods are known in the art such as methods refined for hydrophobic or precipitated proteins, comprising methods using high dilution, addition of detergents, urea etc.
It is within the general knowledge of the skilled person to choose the specific methods according to the criteria outlined above.
It should in this connection here be noted that none of the culture solutions disclosed in WO 97/20921 fulfill these criteria, since the problem of said disclosure had nothing to do with the problem of recovering proteins that were not in solution.
Accordingly, all of the specific culture solutions disclosed in the WO 97/20921 document were culture solutions wherein the protein of interest in the supernatant of the culture solutions had a concentration of more than 80% of the total concentration of the protein of interest in the culture solution as such.
An advantage of the method, as described herein, is tha
Laustsen Mads Aage
O'Reilly Michael John
Simpson Curran Matthew
Garbell Jason I.
Lambiris Elias J.
Novozymes A/S
Saucier Sandra E.
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