Drug – bio-affecting and body treating compositions – Enzyme or coenzyme containing – Stabilized enzymes or enzymes complexed with nonenzyme
Reexamination Certificate
1999-09-30
2002-06-04
Weber, Jon P. (Department: 1651)
Drug, bio-affecting and body treating compositions
Enzyme or coenzyme containing
Stabilized enzymes or enzymes complexed with nonenzyme
C424S094100, C424S094200, C424S094600, C424S094610, C424S094620, C424S094630, C424S094650, C424S094660
Reexamination Certificate
active
06399059
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to a thermally stable enzyme composition and a method of manufacturing the same, and more particularly, to an enzyme composition thermally stable in an aqueous solution and a method of manufacturing the same.
An enzyme can effect a catalytic reaction under milder conditions and exhibits a high substrate specificity, compared with a chemical reaction. Therefore, an enzyme is widely used in the fields of food industry, chemical industry, pharmaceutical industry, etc.
However, the enzyme is generally unstable. Particularly, the enzyme stability is low in a solution. It should be noted that a thermal denaturation begins to take place in many lipid-decomposing enzymes if these enzymes are exposed to temperatures exceeding about 35° C., making it difficult to utilize an enzyme in a reaction system using a substrate having a high melting point. Also, since an enzyme is thermally unstable, a freeze-drying method that is less likely to be affected thermally is utilized mainly for concentrating and preparing an enzyme powder. However, the freeze-drying method requires a tremendous facility investment for the mass production, resulting in a high running cost such as a utility cost.
For improving the thermal stability of the enzyme within an aqueous solution, it has been proposed to add a stabilizer to the enzyme. For example, it has been studied to add an amino acid such as albumin, casein, or sodium glutamate; a reducing agent such as protein, mercapto ethanol, or cysteine; a polyol such as glycerol, sucrose, or sorbitol; or a water-soluble high molecular weight substance such as dextran to an enzyme solution. Also proposed are a method of modifying an enzyme surface with, for example, an emulsifier or another amphoteric substance as disclosed in Japanese Patent Disclosure (Kokai) No. 6-113847, a method of modifying an enzyme surface with an oil-soluble substance having an isoprenoid structure as disclosed in Japanese Patent Disclosure No. 6-269285, and a method of stabilizing an enzyme by dissolving a phospholipid in an alcoholic solvent, followed by mixing the resultant solution with an enzyme and subsequently drying the mixture (Japanese Patent Disclosure No. 1-27719).
However, the lipid-decomposing enzyme obtained by the conventional methods described above is stable only under temperatures of about 37 to 60° C. and, thus, is relatively unstable under heat, making it relatively difficult to use the enzyme for an enzyme synthesis, etc. using a substrate having a high melting point. Also, in the method of preparing an enzyme powder by a spraying method, which is a typical drying method, the enzyme is concentrated under heat and, then, dried within a hot gaseous stream. Therefore, the enzyme activity is lowered.
BRIEF SUMMARY OF THE INVENTION
An object of the present invention is to provide a thermally stable enzyme composition exhibiting an improved heat resistance even within an aqueous solution and capable of stably performing an enzyme reaction under high temperatures.
Another object is to provide a method of preparing an enzyme composition that permits stably preparing an enzyme powder while preventing the enzyme activity from being lowered by the heat in the enzyme powder preparation even by a drying method under heat.
These objects can be achieved according to the present invention by adding a heat stabilizer comprising a combination of an oil-soluble vitamin and a phospholipid to an enzyme solution. The heat stability of the enzyme is significantly improved by adding a heat stabilizer including an oil-soluble vitamin and a phospholipid.
Thus, the present invention provides an enzyme composition comprising an enzyme and a heat stabilizer including a phospholipid and an oil-soluble vitamin.
The present invention also provides a method of preparing a thermally stable enzyme, comprising the step of drying an enzyme solution containing a phospholipid and an oil-soluble vitamin to obtain an enzyme powder. The drying should preferably be performed by a spray drying.
In a preferred embodiment of the present invention, the enzyme composition of the invention contains 0.01 to 200% by weight of a phospholipid and 0.01 to 100% by weight of an oil-soluble vitamin, based on the enzyme weight.
The enzyme used in the present invention should desirably be a lipid-decomposing enzyme. Also, it is desirable to use an oil-soluble vitamin selected from the group consisting of tocopherol, tocotrienol, retinol, calciferol, phylloquinone, and ubiquinone.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a method of preparing an enzyme, in which a phospholipid and an oil-soluble vitamin are added to an enzyme solution to improve the thermal stability of the enzyme and which permits suppressing the deactivation of the enzyme by heat in a powder preparation by a drying method under heating to make it possible to prepare an enzyme powder.
The enzyme used in the present invention, which is not particularly limited, includes preferably a lipid-decomposing enzyme, protease, and sugar-decomposing enzyme. It is particularly desirable to use a lipid-decomposing enzyme. In addition to the enzyme formulations available on the market, the enzyme can be used in the present invention in the form of a microorganism culture solution, a plant extraction liquid and an animal cell extraction liquid. Further, a culture solution, a concentrated extraction liquid and a desalted concentrated solution can also be used as the enzyme in the present invention.
The lipid-decomposing enzyme used in the present invention includes lipases, phospholipases, and esterases. The lipases include, for example, lipoprotein lipase, monoacyl glycerollipase, diacyl glycerollipase, triacyl glycerollipase, and galactolipase. The phospholipases include, for example, lyso phospolipase, and phospholipases A1, A2, B, C and D. Further, the esterases include, for example, choline esterase, cholesterol esterase, pectin esterase, tropine esterase, acetylcholine esterase, acetyl esterase, carboxy esteradse, and aryl esterase.
The sugar-decomposing enzymes used in the present invention include, for example, amylase, glucosidase, cellulase, xylanase, dextranase, chitinase, lysozyme, galactosidase, mannosidase, glucuronidase, hyaluronidase and pectin lyase.
The protease used in the present invention includes endopectidases and exopeptidases. The endopectidases include, for example, acid proteinase, serine protease, cysteine protease, asparagic acid protease, thiol protease, and carboxy protease. On the other hand, the exopetidases include, for example, dipeptidylamino peptidase and dipeptidylcarboxy peptidase.
The microorganisms producing the enzymes used in the present invention, which are not particularly limited, can be selected from bacteria, yeasts, filamentous viruses, and actinomycetes, and include, for example, Psudomonas species, Alcaligenes species, Arthrobacter species, Staphylococcus species, Torulopsis species, Escherichia species, Micotorula species, Propionibacterum species, Chromobacterum species, Xanthomonas species, Lactobacillus species, Clostridium species, Candida species, Geotrichum species, Sacchromycopsis species, Nocardia species, Fuzarium species, Aspergillus species, Penicillium species, Mucor species, Rhisopus species, Phycomycese species, Puccinia species, Bacillus species and Streptmycese species.
A culture medium containing soybean powder, peptone, corn steep liquor, K
2
HPO
4
, (NH
4
)
2
SO
4
, MgSO
4
.7H
2
O, etc. can be used for growing the microorganisms given above. It is. desirable for the culture medium to contain soybean powder in an amount of 0.1 to 20% by weight, preferably 1.0 to 10% by weight. Peptone should desirably be contained in an amount of 0.1 to 30% by weight, preferably 0.5 to 10% by weight. Corn steep liquor should desirably be contained in an amount of 0.1 to 30% by weight, preferably 0.5 to 10% by weight. K
2
HPO
4
should desirably be contained in an amount of 0.01 to 20% by weight, preferably 0.1 to 5% by weight. (NH
4
)
2
SO
Endou Yoriko
Minoshima Ryouichi
Bacon & Thomas
Patten Patricia
The Nisshin Oil Mills Ltd.
Weber Jon P.
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