Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Oxidoreductase
Reexamination Certificate
1998-10-16
2002-03-12
Weber, Jon P. (Department: 1651)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Oxidoreductase
C435S267000
Reexamination Certificate
active
06355461
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to substantially anhydrous (i.e. substantially water-free) liquid compositions comprising: 1) an enzyme; 2) a substance which (a) in aqueous medium is a substrate for the enzyme, (b) in aqueous medium is a precursor for a substrate for the enzyme, or (c) is a cofactor for the enzyme; and 3) a non-aqueous liquid phase.
The invention is well suited to a wide variety of both non-food and food applications.
BACKGROUND OF THE INVENTION
Industrial enzymes have generally been formulated as particulate solids (e.g. in powder or granulated form, optionally with a coating of some kind) or in the form of a water-based solution. A number of solid formulations (e.g. enzyme powders) have the disadvantage that dust formation readily takes place, which—unless special precautions are taken—may result in contamination of the surrounding environment and thereby pose a risk to the health of persons handling such formulations.
While the use of water-based, liquid enzyme formulations essentially eliminates risk of dust formation, owing to the fact that practically all enzymes exert their activity in the presence of water it is generally not feasible to prepare storage-stable formulations of this type which incorporate—in one and the same composition—a free (e.g. unencapsulated or uncoated) enzyme and, for example, a substance which is a substrate for the enzyme (including in this connection an enzyme substrate which, via the agency of the enzyme, and normally in combination with one or more further reactants or reagents, reacts to form a species which undergoes a further subsequent reaction of importance in connection with the particular purpose for which the liquid enzyme formulation is intended).
By way of example, in connection with the use of a peroxidase in conjunction with an oxidizable substance known as a “mediator” (also known as an “enhancer” or an “accelerator”)—e.g. a mediator of the substituted phenothiazine or substituted phenoxazine type—for the purpose of “dye-transfer inhibition” (i.e. inhibition of fabric-to-fabric transfer of dye) in connection with washing of dyed fabric or textiles (see, e.g., WO 94/12621), or for bleaching fabrics, e.g. denim fabrics (as described in WO 96/12845 and WO 96/12846), it will generally not be possible to include the peroxidase and the mediator (and, optionally, a source of hydrogen peroxide) in one and the same, water-based, liquid composition without rapid oxidation, and subsequent transformation, of the mediator taking place.
With reference again, by way of example, to the above-mentioned bleaching of fabric (such as denim fabric), when employing currently available liquid enzyme formulations the relatively short time span within which it is possible to operate once the enzyme (e.g. a peroxidase) has been brought into contact with the mediator will often necessitate (i) the establishment of separate containers or vessels (tanks or the like) each containing one of the interacting components, and from which the individual components can be dosed to the medium (aqueous medium) in which the bleaching process is to take place, and (ii) dosing of the individual components from the respective containers to the medium in the correct amounts and correct relative proportions. Thus, not only are multiple containers required, but it is also necessary to perform multiple unit operations (including multiple dispensing and dosing operations).
There is thus a need for liquid, storage-stable compositions which (a) contain not only an enzyme, but also a substance (e.g. a substance of one of the types mentioned earlier, above) which otherwise—in the presence of water and via the agency of the enzyme—will undergo a reaction of interest, and (b) are suited for subsequent introduction into or contact with an aqueous medium, thereby initiating the reaction in question. The present invention provides compositions fulfilling this need.
DETAILED DESCRIPTION OF THE INVENTION
The present invention thus relates to a substantially water-free liquid composition comprising:
(A) an enzyme;
(B) a substance (i.e. one or more substances) selected from (i) substances which in aqueous medium are substrates for said enzyme, (ii) substances which in aqueous medium are precursors for substrates for said enzyme, and (iii) substances which are cofactors for said enzyme; and
(C) a non-aqueous liquid phase.
The term “substantially water-free” as employed in the present specification and claims in connection with a liquid composition of the invention is intended to indicate that the water content of the composition [normally to be understood as the content of free (unbound) water in the composition] is sufficiently low to ensure a thermodynamic activity of water in the composition which is low enough to hinder the onset of interaction between, on the one hand, the enzyme(s) in question and, on the other hand, the substrate(s), substrate precursor(s) and/or cofactor(s) in question.
The acceptable upper limit for the water content of a given type of embodiment of a composition according to the invention will depend, inter alia, on the nature of the enzyme, the nature and properties of the enzyme substrate, enzyme substrate precursor or enzyme cofactor, and the nature and properties of the non-aqueous liquid phase. For some types of embodiments of compositions of the invention, it may be possible to prepare satisfactorily stable compositions having a water content of about 5% w/w, or possibly even higher. However, a water content not exceeding about 2% w/w is normally to be preferred.
For numerous embodiments of compositions of the invention (e.g. compositions of the types exemplified and tested in the working examples herein), a more preferable upper limit for water content will normally be in the vicinity of 1% by weight (w/w) of the composition. It will, however, generally be desirable that the water content of many types of embodiments does not exceed about 0.8% w/w, more desirably about 0.6% w/w, and a very desirable upper limit will often be about 0.5% w/w. For certain embodiments, an upper limit for the water content of about 0.2% w/w may be appropriate in order to achieve adequate stability of the composition.
Water content may suitably be determined, for example, by means of so-called Karl Fischer titration [see, e.g., J. S. Fritz and G. H. Schenk, Jr.,
Quantitative Analytical Chemistry,
2nd edition, Allyn and Bacon, Inc., Boston (1969), p. 276, and reference given therein].
Preparation of Compositions of the Invention
With regard to the preparation of a composition according to the invention, a number of approaches are applicable, depending mainly on the form in which the enzyme(s) to be incorporated therein are initially available: If the enzyme(s) is/are available in the form of a substantially water-free solid preparation, the solid enzyme preparation (optionally together with other components of the composition, particularly components which are insoluble, or are at least of low solublility, in the liquid phase employed) may—subsequent to any comminution step which may be necessary to achieve appropriately sized solid particles—simply be dispersed, where appropriate in conjunction with the addition of one or more suitable dispersing agents, in the non-aqueous liquid phase in question by methods known per se.
In cases where the enzyme(s) is/are available as an aqueous solution or concentrate, it is often possible to add to the solution/concentrate a non-aqueous—often water-immiscible—liquid substance (e.g. a paraffin oil or the like) which is inert towards the components of the solution/concentrate and forms an azeotrope with water, and which therefore—under appropriate temperature and pressure conditions—may be used to remove water from the solution/concentrate by distillation (see, e.g., EP 0 696 315). In such cases it may be appropriate to add, portionwise at intervals during the course of the distillation process (and, optionally, at the beginning of the distillation process), another non-aqueous liquid
Henriksen Lotte Rugholm
Lykke Mads
Garbell Jason I.
Lambris Elias M.
Novozymes A/S
Weber Jon P.
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