Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Stablizing an enzyme by forming a mixture – an adduct or a...
Reexamination Certificate
1999-12-03
2002-06-18
Weber, Jon P. (Department: 1651)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Stablizing an enzyme by forming a mixture, an adduct or a...
C435S183000, C435S184000, C424S094300
Reexamination Certificate
active
06406897
ABSTRACT:
BACKGROUND OF. THE INVENTION
1. Field of the Invention
The present invention is related to a protein modified to improve its stability and a method for producing the same. More particularly, the present invention is related to a protein coupled to &bgr;-1,3-glucan branched with &bgr;-1,6-linkage, which shows an improved stability while retaining its activity, to a method for the production thereof, and to a composition for external or topical application comprising the stabilized proteins.
2. Description of the Related Arts
Proteins play various roles in living body, for example as an biocatalyst in the metabolisms, as signal transmitting agents, or the like and are commonly used advantageously in a variety of applications such as laundry industry (e.g. detergents), cosmetic industry, pharmaceutical compositions (e.g. digestants, anti-inflammatory drugs and the like), food industry (e.g. meat tenderizing agents) and so on.
However, their uses have been limited due to their instability. antigenicity, or other safety problems. In fact, in addition to skin irritation, unsatisfactory effectiveness on the skin and/or difficulty in the production of the external or topical application compositions such as cosmetic or dermatological compositions incorporated with the proteins, the short shelf-life has restricted the practical use of these compositions.
Several attempts such as immobilization or chemical modification have been made to improve the stability of the proteins so far. Although these attempts did not provide a satisfactory result, representative examples thereof are explained below.
U.S. Pat. No. 4,556,554 teaches a cosmetic composition for removal of sebum exudate from the skin, which comprises immobilized enzymes in cosmetically acceptable vehicles, and the enzymes are immobilized to functional polymers in known manners by chemical and or physical means. They are released from immobilization upon application to the skin. However, this immobilization does not provide a satisfactory improvement of stability of the enzymes in the composition.
Masunaga et al (T. Masunaga et al., IFSCC, Yokohama, A205, pp 483-501) reports proteases which are chemically coupled to polyethylene glycol. The proteases show improved stability and reduced skin-irritation. But they still have drawbacks that their shelf-time is not sufficiently prolonged and their preparation is complex.
U.S. Pat. No. 5,230,891 assigned to Kanebo Limited teaches a modified protease and their production method. Polysaccharide such as dextran, alginic acids or carrageenin is reacted with cyanuric trichloride to give a triazine ring-bound polysaccharide, which is reacted with protease to give a protease coupled to polysaccharide via triazine ring. This method still has a drawback that it is very complicated.
EP 0,803,257 A2 and JP 4-141097A teach an enzyme stabilization by oxidative process using polysaccharides. Thus-stabilized enzymes can, for example, be attached to surfaces of medical devices to minimize undesirable biological reaction associated with medical devices or attached to the surface of the reactor. However, it is not certain the stabilized enzymes can be incorporated into cosmetic or dermatological compositions
Therefore, there has been a need to provide proteins so stabilized so as to be incorporated into compositions for external or topical applications.
SUMMARY OF THE INVENTION
The present invention provides a modified protein, which has an improved stability, said protein being coupled to &bgr;-1,6 residue branched from &bgr;-1,3-glucan.
The present invention further provides a method for producing the modified protein, which comprises the steps of
(a) reacting a glucan with periodates to oxidize &bgr;-1,6 residue of the glucan into aldehyde;
(b) removing unreacted periodates from the reaction solution;
(c) adding to the reaction solution a protein in an amount of 0.00001~10.0% by weight and allowing the protein to contact with the oxidized glucan; and
(d) adding to the reaction solution a reducing agent in an amount of 0.0001~1.0% by weight.
The method may further comprise the step of washing the product obtained in the step (d).
The present invention still provides a composition for external or topical application, which comprises the modified protein as an active ingredient.
The present invention will be described in detail below.
DETAILED DESCRIPTION OF THE INVENTION
According to the present invention, the protein is stabilized by coupling to &bgr;-1,3-glucan regularly branched with &bgr;-1,6-linkage (herein after referred to simply as “glucan”). The glucan has a neutral pH in aqueous phase, has a triple helix shape contributing to its stability, and can be modified only at its &bgr;-1,6-residue by chemical means. In contrast, polysaccharides such as dextran are modified randomly or at every residue. Therefore, glucan allows a unique modification of proteins in terms of coupling index between glucan and protein, and stability and activity of the modified protein.
The glucan employed according to the invention may include schizophyllan originated from
Schizopyllum commune
, scleroglucan from Sclerotinia sp., lentinan from
Lentinus edodes
. Other glucans can be employed as long as they have &bgr;-1,3-chain branched with regular &bgr;-1,6-linkage.
The glucans as naturally occurred have usually a molecular weight ranging from several hundred thousands to several millions. They can be used as they are, or treated by mechanical or chemical means to have a molecular weight from about 50,000 to several hundred thousands. For example, microfluidizer. sonicator or &bgr;-glucanase treatment can be used to break the intact glucan to give a smaller molecular weight glucans. The term of “glucan” is used herein to include the intact glucans as well as the small molecular weight glucans.
The glucans may be used in an aqueous solution having a concentration of 0.001~20.2% by weight, and preferably 0.1~5.0% by weight.
The proteins which can be modified to improve their stability according to the present invention may include, but not limited thereto, proteases such as papain, bromelain, ficin, trypsin, chymotrypsin, collagenase, elastase, plasmin or bacterial protease; carbohydrate-lysis enzymes such as amylase, glucoamylase, cellulase, pectinase, xylanase, &agr;-glucosidase, &bgr;-glucosidase, &bgr;-galactosidase, &bgr;-galactosidase, &bgr;-glucanase, chitinase or mannanase; lipases such as phospholipase or triacylglycerol hydrolase; nucleic acid lysis enzymes such as DNase or RNase; phosphatases; lysozymes; catalases; superoxide dismutases; transglutamninases, peroxidases; photolyases; complex enzymes originated from microorganisms; cytokines; growth factors; hormones; antigens; antibodies; immunoglobulins; lactoferrins; metallothioneins; thioredoxins; antimicrobial proteins; or antioxidative proteins;. The proteins also include active peptides thereof or conjugates with saccharides or lipids. They may be used singly or in combinations thereof.
The method for the production of the modified proteins will be described in detail hereinafter.
The method comprises the steps of (a) reacting a glucan with periodates to oxidize &bgr;-1,6-linkage of the glucan into aldehyde; (b) removing unreacted periodates from the reaction solution; (c) adding to the reaction solution a protein in an amount of 0.00001~10.0% by weight and allowing the protein to contact with the oxidized glucan; and (d) adding to the reaction solution a reducing agent in an amount of 0.0001~1.0% by weight. The method may further comprise the step of washing the desired product obtained in the step (d).
The step (a) is to oxidize &bgr;-1,6-residue of the glucan into aldehyde, which allows a coupling between the glucan and the protein. Periodates are used in an amount of 0.1~50.0 molar equivalents, and preferably 2~5 molar equivalents with respect to one mole of glucan. The reaction mixture is allowed to react in the dark with stirring. Periodates may include, but not limited to, HIO
4
, H
5
O
6
, NaIO
4
, Na
3
H
2
IO
4
or KIO
4
. After the reaction, e
Kang Byung Young
Kim Mu Sung
Lee Dong Chul
Lee Sung Gu
Guttman Harry J
Pacific Corporation
Weber Jon P.
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