Precifitation process for exocellular proteins

Details Precifitation process for exocellular proteins Precifitation process for exocellular proteins

1993-05-26

1995-09-26

Cintins, Ivars

Liquid purification or separation

Processes

Liquid/liquid solvent or colloidal extraction or diffusing...

210650, 210665, 210666, 210669, 210694, 435183, 435815, B01D 1500

Patent

active

054532000

DESCRIPTION:

BRIEF SUMMARY
FIELD OF THE INVENTION

This invention relates to a process for the purification and separation of exocellular proteins from fermenter broths. More particularly, the invention relates to a process for separating enzymes in solid form, coloring and odor-emitting substances being removed.


STATEMENT OF RELATED ART

Numerous enzymes, especially hydrolases, such as for example proteases, amylases or lipases, are produced by fermentation of microorganisms. Suitable microorganisms and processes for their production are described, for example, in the following patents and patent applications: DE 18 00 508, DE 22 24 777, DE 25 51 742, U.S. Pat. No. 3,827,938, WO 88/01293, DE 18 07 185, U.S. Pat. No. 3,740,318, DE 23 34 463, DE 20 26 092, EP 0 232 169, EP 0 220 921, EP 0 247 647 and EP 0 246 678.
Strongly coloring or strong-smelling impurities are unacceptable for numerous applications, for example for the use of the enzyme solutions in liquid detergents. Accordingly, in the industrial production of the enzymes, the impurities tend to be removed by precipitation processes. However, hitherto known precipitation processes have the disadvantage that considerable losses of yield have to be accepted in order to obtain good color quality. To counteract these difficulties, German patent application P 39 11 099.0 describes a precipitation process in which a masking agent is added to an enzyme solution produced by fermentation and a precipitate is subsequently prepared by adding two water-soluble, mutually precipitating ionic compounds in any order and optionally introducing other adsorbents, for example active carbon.
According to German patent application DE 38 21 151, fermenter broths and/or enzyme solutions are provided with reducing additives in order to reduce odor emission and to improve color quality.
A similar process is described in German patent application P 39 30 284.9, according to which cells of fungi, plants and/or bacteria or cell wall fragments of the above-mentioned organisms are added as selective adsorbents to a fermenter broth. German patent application P 39 15 277.4 also describes a similar process in which an acidic aqueous solution of an aluminum salt and, optionally, additional precipitants are added to the enzyme solution above a pH value between 5 and 11, water-soluble constituents are removed and a masking agent, such as an acid of boron or the like, is added after precipitation.
All the above-mentioned processes are based on the idea of binding the troublesome constituents to the surface of an adsorbent or co-precipitating them with the precipitate of an adsorbent produced in situ, so that an enzyme solution of improved purity remains behind. Although very pure enzyme solutions can be obtained by these processes, the enzyme yield naturally decreases with increasing purification, so that a compromise always has to be made between good quality and good quantity.
It is already known that proteins can be separated from fermenter solutions by precipitation of the proteins themselves rather than the troublesome impurities. However, where the usual precipitants are used in the usual concentrations, the impurities are co-precipitated so that the required purity cannot be obtained in this way.


DESCRIPTION OF THE INVENTION



SUMMARY OF THE INVENTION

The present invention is based on the surprising observation that the concentration precipitation of proteins, particularly hydrolases, is possible when certain substances evidently omnipresent in fermenter broths, which prevent concentration precipitation, are removed by a preliminary treatment with an adsorbent.
Accordingly, the present invention relates to a process for the separation of exocellular proteins of microorganisms from a filtered fermenter broth, characterized in that, in a first step, substances which impede precipitation of the proteins are removed by means of a solid adsorbent, the remaining solution is concentrated to a protein content of around 30 to 40% by weight and the protein is subsequently precipitated at pH values of 6 to 10 an

REFERENCES:
patent: 3101302 (1963-08-01), Inglett
patent: 3711462 (1973-01-01), Abdo
patent: 3740318 (1973-06-01), Churchill et al.
patent: 3827938 (1974-08-01), Aunstrup et al.
patent: 4007115 (1977-02-01), Howe
patent: 4886602 (1989-12-01), Kuehne et al.
Chemical Abstracts, vol. 107, No. 15, Oct. 12, 1987, Columbus, Ohio; Abstract No. 129968D, "Purification of an Enzyme Concentrate", p. 345; Col. L (See Abstract).
Chemical Abstracts, vol. 87, No. 1, Jul. 04, 1976, Columbus, Ohio; Abstract No. 4003x, I Alemzadeh et al.: "bacterial flocculation with sodium bentonite", p. 334; Col. R; (See Abstract).

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