Process for producing a stable carboxylesterase in dry form

Details Process for producing a stable carboxylesterase in dry form Process for producing a stable carboxylesterase in dry form

1997-01-14

1998-08-11

Weber, Jon P.

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Stablizing an enzyme by forming a mixture, an adduct or a...

435197, C12N 996, C12N 918

Patent

active

057926360

DESCRIPTION:

BRIEF SUMMARY
TECHNICAL FIELD

The present invention relates to a process for producing a stable carboxylesterase in dry form. The dry enzyme composition is added to food and drink (e.g., bread, brewed seasonings, liquors, processed meat products) during the production process to thereby impart thereto a good aroma (ester aromas).


BACKGROUND ART

Heretofore, various substances are used in order to stabilize enzymes. For example, known are saccharides (e.g., lactose, glucose, saccharose, dextrin, arabitol, sorbitol, mannitol, inositol, .beta.-cyclodextrin), amino acids and chelating agents (Japanese Published Unexamined Patent Application No. 34001/80) as stabilizers for sarcosine oxidase; lactose, sucrose, gelatin, aspartic acid and glutamic acid (Japanese Published Unexamined Patent Application No. 134,991/83) as stabilizer for serratiopeptidase; and sucrose, glucose, glycerol, gelatin, albumin, amino acids, etc. ("Freeze-Drying, and Protecting Substances" edited by Tokio Nei, published by Tokyo University Publishing Co., Mar. 20, 1972), and also ethylene glycol, glycerol, sorbitol, inositol, glucose, sucrose, etc. protecting enzyme proteins.
As a laboratory reagent grade carboxylesterase (EC. 3.1.1.1), known is a carboxylesterase in liquid form derived from porcine liver such as (1991)!.
Additionally known is a freeze-dried product of rabbit liver-derived (1993)! which is not satisfactorily stable. The analysis of this freeze-dried enzyme as carried out in accordance with the method described in "Chemistry of Saccharides (last volume), p. 329, published by Tokyo Kagaku Dojin" has revealed that any stabilizer including saccharides is not added thereto.
Carboxylesterases derived from animal organs are known not only in the laboratory use but also for industrial use, which is used only in liquid form(see W093/09681).


DISCLOSURE OF THE INVENTION

The present invention relates to a process for producing a stable carboxylesterase in dry form, which comprises adding a saccharide to a solution containing carboxylesterase and drying the solution.
Carboxylesterase to be used in the present invention may be derived from animal organs, especially porcine, bovine, ovine or caprine organs such as liver, kidney and heart.
One embodiment of the process of the present invention for producing a stable carboxylesterase in dry form from any of such animal organs is as follows:
After an animal organ is minced, a buffer (pH 6 to 7) containing sucrose is added to the minced organ and the minced organ is shattered and then centrifuged. The resulting supernatant is adjusted to have a pH of 4.5 to 5.5 with an acid (e.g., acetic acid) and then again centrifuged to obtain a precipitate. This precipitate is defatted with a solvent (e.g., acetone) and suspended in a buffer (pH 6 to 7). Ammonium sulfate is added to the resulting suspension at 70% saturation, and centrifuged. The resulting precipitate is suspended in 3.2 M ammonium sulfate to obtain an enzyme suspension. A saccharide is added to this enzyme suspension directly thereto or after the enzyme suspension has been diluted with, for example, water or the like or after it has been dissolved through desalting. Thus is prepared a solution of the enzyme, which is then dried.
The saccharide to be added to the enzyme liquid may be at least one selected from maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose, glucose, sucrose, trehalose, lactose, fructose, maltoheptaose, maltitol, maltotriitol, maltotetraitol, inositol, sorbitol and lactitol, preferably at least one selected from maltose, sucrose, maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, maltitol, maltotriitol, maltotetraitol and lactitol, more preferably maltose or maltitol.
The amount of the saccharide to be added may be 0.1 to 30% w/v, preferably 0.1 to 20% w/v, relative to the enzyme solution.
The drying may be conducted according to any of vacuum freeze-drying, spray-drying, aeration-drying or the like. The vacuum freeze-drying may be conducted at a vacuum degree of 130 Pa or lower, at a freezi

REFERENCES:
patent: 4904592 (1990-02-01), Freeman et al.
van de Beek et al. (1969) Neth. Milk Dairy j., 23, "Preservation of the Enzymatic Activity of Rennin During Spray Drying and During Storage, and the Effect of Sugars and Certain Other Additives", pp. 46-54.
Back et al. (1979) Biochemistry, 18(23), "Increased Thermal Stability of Proteins in the Presence of Sugars and Polyols", pp. 5191-5196.
Arakawa et al. (1982) Biochemistry, 21(25), "Stabilization of Protein Structure by Sugars", pp. 6536-6544.
Gekko (1982) J. Biochem., 91, "Calorimetric Study on Thermal Denaturation of Lysozyme in Polyol-Water Mixtures", pp. 1197-1204.
Schmid (1979) Adv. Biochem. Eng., 12, "Stabilized Soluble Enzymes", pp. 42-118.

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