Stabilized lactoperoxidase and glucose oxidase concentrate

Details Stabilized lactoperoxidase and glucose oxidase concentrate Stabilized lactoperoxidase and glucose oxidase concentrate

1999-10-25

2001-11-06

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

Reexamination Certificate

active

06312687

ABSTRACT:

This invention relates to a stabilized enzyme concentrate of glucose oxidase and lactoperoxidase.
W091/11105 discloses anti-microbial compositions which contain iodide and hiocyanate anions, glucose oxidase, D-glucose and lactoperoxidase. These compositions have excellent anti-microbial properties and are effective against bacteria, yeasts and moulds. It is further disclosed that the compositions may be provided in concentrated substantially non-reacting form. Such concentrated compositions maintain physical separation of the glucose oxidase and at least one of its substrates, namely D-glucose, water and oxygen, such that H
2
O
2
production is substantially prevented during storage. It is also disclosed that the concentrated compositions may incorporate at least one buffering agent to minimize the fall of the pH which may otherwise occur after activation of the concentrated composition. However, although a very successful product exists in which D-glucose, sodium thiocyanate and potassium iodide are provided in a substrate solution and the lactoperoxidase and glucose oxidase are provided in a concentrated enzyme solution, there exists a problem with the enzyme solution regarding its comparatively short shelf-life which is around twelve weeks at ambient temperature. Therefore, there is a need to provide an enzyme solution with a much longer shelf-life.
It is known that the stabilization of an enzyme in solution is difficult. W090/05182 describes some of the attempts which have been made to overcome this problem, for example the addition of sugars or glycerol to enzyme solutions or by freeze drying. W090/05182 states that freeze drying is expensive and often results in denaturation and then discloses a method of protecting proteins against denaturation on vacuum or air drying which comprises mixing an aqueous solution of the protein with a soluble cationic polyelectrolyte and a cyclic polyol, and removing the water from the solution.
A method of stabilizing proteins in solution is disclosed in W095/10605 in which a protein stabilizer additive which comprises two or more of a tris compound of formula I: (HOCH
2
)
3
—C—R, wherein R is various groups for example C
1-4
alkyl which may be optionally substituted, a polvelectrolyte; a buffer and one or more further components for example, divalent metal salts. On page 3 of this document it is disclosed that “The said further component may be selected from the group comprising divalent metal ions, chelators, for example EDTA, EGTA or citrate (not with peroxidases) or polyols”. The statement would direct the person skilled in the art, faced with the problem of formulating a stable enzyme concentrate containing lactoperoxidase, away from the use of citrates. It would point him instead to the use of non-chelating buffers, for example BIS/TRIS.
It will be appreciated that, given the known difficulties of stabilizing one enzyme in solution, the problem is further compounded when a stable mixture of two enzymes in solution is required since each enzyme has its own optimum requirements which may not be compatible with the optimum requirements of the other enzyme. In addition, it is not only required that the enzyme concentrate is chemically stable but it must also be preserved against microbial attack if it is to be used in anti-microbial compositions. There may be incompatibility between the agent(s) required to produce chemical stability and the agent required to produce preservation.
The present invention provides a stabilized aqueous enzyme concentrate composition which comprises:
a) 1000 to 1800 units/ml of lactoperoxidase;
b) 1500 to 2750 units/ml of glucose oxidase;
c) 10 to 20% w/v of an alkali metal halide salt; and
d) a chelating buffering agent present in an amount such that the pH of the composition is in the range of 5.5 to 6.5.
Although it is known that sodium chloride may be used to preserve lactoperoxidase, for example at a level of 1.8% or 12%, it is nevertheless surprising that a concentrate of lactoperoxidase and glucose oxidase may be stabilized chemically and microbially preserved for long periods by a combination of a chelating buffering agent and an alkali metal salt.
WO95/26137 (page 43) discloses the effect of pH on the anti-microbial activity of final compositions which have been prepared by combining and diluting two concentrates (Phase A and Phase B) to 0.9% and 0.05%.
Component
Concentration
Phase A
(w/v %)
D-glucose
45 to 55
Sodium thiocyanate
0.42 to 0.52
Potassium iodide
0.66 to 0.80
Phase B
Lactoperoxidase
5,500 Units/ml
Glucose oxidase
2,250 Units/ml
The pH of each phase was adjusted to between 5.5 and 6.5 with buffer solutions. The pH of each combined concentrate mixture after dilution was adjusted using a citrate/phosphate buffer. However, this document does not disclose or suggest the unexpected and beneficial effect of obtaining an enzyme concentrate which is stable over a prolonged period.
EP 307,376 and WO91/11105 disclose diluted anti-microbial compositions which contain citrate salts and/or sodium chloride. However, there is no disclosure of a stabilised enzyme concentrate according to the present invention.
EP 252,051 discloses a method of stabilizing lactoperoxidase in milk products, foodstuffs and pharmaceuticals to which the enzyme lactoperoxidase has been added, wherein the product containing lactoperoxidase is adjusted with regard to pH, so that the pH is in the range 3.25 to 6 at the dissolution in water. It does not disdose a stabilised enzyme concentrate according to the present invention.
Suitably, the enzyme concentrate is stable for at least 6 months. Preferably the enzyme concentrate is stable for at least 9 months. More preferably the enzyme concentrate is stable for at least 12 months. Most preferably the enzyme concentrate is stable for at least 18 months.
The stability of the enzyme concentrate is determined by assaying the activity of the lactoperoxidase and glucose oxidase at regular intervals using standard assay techniques which are given in the examples of this specification. The efficacy of antimicrobial preservation is assessed using the British Pharmacopeia test as given in the 1993 volume, Appendix XVIC A191.
Suitably the stability of the enzyme concentrate is such that the activities of lactoperoxidase and glucose oxidase are maintained at at least 60% of their original activity over the time period examined. Preferably the activities of lactoperoxidase and glucose oxidase are maintained at least 75% of their original activity after storage for 6 months at 25° C. More preferably the activities of lactoperoxidase and glucose oxidase are maintained at least 75% of their original activity after storage for 12 months at 25° C.
Preferably the alkali metal halide is selected from sodium chloride, potassium chloride, sodium bromide, potassium bromide, sodium iodide or potassium iodide or mixtures thereof. More preferably the alkali metal halide is sodium chloride.
Preferably the concentration of the alkali metal halide is in the range of 12 to 18% w/v. More preferably the concentration of the alkali metal halide is in the range of 14 to 16% w/v. Most preferably the concentration of the alkali metal halide is in the range of 14.5 to 15.5% w/v.
Suitably the chelating buffering agent is selected from one or more of the following: alkali metal salts of citric, phthalic, tartaric, adipic or succinic acid. Preferably the chelating buffering agent is trisodium citrate. Suitably the amount of chelating buffering agent present is that required to give a final pH of in the range 5.5 to 6.5. Preferably the amount of buffering agent is such that the pH of the composition is in the range 5.7 to 6.2. Most preferably the amount of buffering agent present is such that the pH of the composition is in the range of 5.9 to 6.1. Optionally monosodium orthophosphate may be present in an amount ranging from 0.05 M to 0.5 M, preferably 0.25 M. Preferably the weight of trisodium citrate used is in the range 0.5 to 1.5% w/v.


REFERENCES:
patent: 5453284 (1995-09-01), Pellico
patent: 88/02600 (1988-04-01)

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