EP0669975A1

EP0669975A1 - Stabilization of hemoproteins

Info

Publication number: EP0669975A1
Application number: EP93924531A
Authority: EP
Inventors: Kaj André HOLM; Niels Krebs Lange
Original assignee: Novo Nordisk AS
Current assignee: Novo Nordisk AS
Priority date: 1992-10-28
Filing date: 1993-10-28
Publication date: 1995-09-06
Also published as: WO1994010298A1; DK131792D0; JPH08502411A

Abstract

Hemoproteins are stabilized in aqueous solutions by means of a stabilizer of the formula (I): R-O(CHR'-CH2-O-)nH, wherein R = H or C1-C20 alkyl, aryl or alkylaryl, R' = H or CH3, and n = 4-50000.

Description

STABILIZATION OF HEMOPROTEINS

TECHNICAL FIELD

This invention relates to the stabilization of hemoproteins, particularly oxidoreductases such as peroxidase or catalase.

BACKGROUND ART

Various oxidoreductases such as peroxidase or catalase are known to be hemoproteins. They can be obtained from many sources, e.g. from horseradish and microorganisms such as Coprinus (EP 179,486), Humicola or Scytalidium (WO 92/17571). Peroxidase can be added to a detergent together with a hydrogen peroxide precursor to improve the bleaching of stains (WO 89/09813) or to inhibit the transfer of dye from one textile to another (WO 91/05839). Other known uses of peroxidase are in the pulp and paper industry (EP 418,201 , EP 429,422, EP 433,258) and in production of particle boards (US 4,432,921). Catalase may be used for removing residual hydrogen peroxide after bleaching, e.g. of textiles. It is in some cases desirable to improve the stability of hemoproteins against denaturation and loss of enzyme activity, and it is the object of this invention to provide a method of stabilizing hemoproteins in solution.

STATEMENT OF THE INVENTION

We have surprisingly found that certain alkoxylated non-ionic compounds, such as polyethylene glycol and alkylphenol ethoxylate, have a strong stabilizing effect on hemoproteins such as peroxidase in solution.

US-A-4,414,326 and EP-A-0 272 578 describe the use of alkoxylated compounds of the above type in an enzymatic reagent composition for the determination of cholesterol. The compositions contained cholesterol esterase, cholesterol oxidase and peroxidase. Said documents describe activation and stabilization of cholesterol esterase and activation of cholesterol oxidase (US-A- 4,414,326 at col. 9 I. 26-28; EP-A-0 272 578 at col. 2 I. 18-20). No effect of the alkoxylated compound on peroxidase was described. Use of cholesterol oxidase and cholesterol esterase is not contemplated in the present invention.

Accordingly, the invention provides a method of stabilizing a hemoprotein in an aqueous solution, characterized by incorporating a stabilizer of the formula:

R-0-(CHR'-CH₂-0-)_nH (I) wherein R = H or C^C^ alkyl, aryl or alkylaryl

R' = H or CH₃, and n = 4-50000.

The invention also provides a process comprising addition of peroxidase and hydrogen peroxide or a precursor thereof, characterized by incorporating said stabilizer. Finally, the invention also provides a peroxidase preparation for use in said bleaching composition or said process, in the form of a slurry, a non-dusting granulate or a stabilized liquid, characterized by comprising said stabilizer.

DETAILED DESCRIPTION OF THE INVENTION

Hemoprotein

The method of the invention may be applied to any hemoprotein, e.g. an oxidoreductase. Examples are peroxidase (EC 1.11.1.7), e.g. from Coprinus (particularly C. cinereus, vide EP 179,486), lignin peroxidase (ligninase), e.g. from Phanerochaete chrysosporium or catalase, e.g. from Humicola or Scytalidium. Other examples are haloperoxidases, i.e. peroxidases that utilize halide ions and hydrogen peroxide to form carbon-halogen bonds in substrates, e.g. chloroperoxidase (EC 1.11.1.10), bromoperoxidase or iodoperoxidase (EC 1.11.1.8). The hemoprotein may be produced by recombinant DNA technology (WO 92/16634). Other examples are cellobiose oxidase or cytochrome oxidase

The amount of peroxidase typically corresponds to 0.1 - 5 PODU per I of aqueous solution. PODU (Peroxidase Unit) is a measure of peroxidase activity defined below.

In the case of peroxidase, the invention provides increased activity, in addition to increased stability.

Stabilizer

The stabilizer used in the invention has the general formula: R-O-(CHR'-CH₂-O-)_nH (I) wherein R = H or 0,-0^ alkyl, aryl or alkylaryl R' = H or CH₃, and n = 4-50000.

The stabilizer may be polyethylene glycol or polypropylene glycol (i.e. R = H in the above formula). In this case, the chain length (n) is preferably 50-500, particularly 100-300.

Alternatively, the stabilizer may be alcohol ethoxylate or alkyl-phenol ethoxylate (propoxylate), where R is a C₁₀-C₁₈ linear or branched alkyl or alkyl-phenyl group. The chain length (n) is preferably 5-50, particularly 5-20. For adequate stabilization, the amount of said stabilizer should be such that the weight ratio of stabilizer to hemoprotein is 1-50.

Suitable amounts of stabilizer are 0.1-5 g/l of aqueous solution, 1-30% by weight of bleaching composition or 1 -10% by weight of peroxidase preparation. A suitable ratio of stabilizer and peroxidase corresponds to 0.2-5 mg of stabilizer per PODU of peroxidase activity (unit defined below).

The stabilizer is typically used at pH in the range 4 - 10, more preferably 6 - 8. Hydrogen peroxide source

In this invention, the source of hydrogen peroxide can be hydrogen peroxide itself, a precursor thereof or an enzymatic system capable of generating it. The precursor of hydrogen peroxide may be an inorganic or organic peracid or salt thereof, e.g. sodium perborate or sodium percarbonate. The enzymatic system may be a combination of a suitable oxidase and a substrate thereof, e.g. glucose and glucose oxidase.

Process using peroxidase or catalase

According to the invention, peroxidase or haloperoxidase may be used together with hydrogen peroxide or a precursor thereof e.g. in a process for bleaching of pulp for paper production (SE 88/0673, EP 418,201), in treatment of waste water from pulp production (US 4,623,465, JP-A 2-31887), in production of mechanical pulp from a fibrous product (EP 429,422, EP 433,258) and for lignin modification, e.g. in particle board production (US 4,432,921). Catalase may be used to remove hydrogen peroxide from fabric which is bleached by an alkaline hydrogen peroxide treatment before dyeing (GB 2,216,149; JP 2-104781).

Peroxidase preparation

The peroxidase preparation of the invention may be provided as a slurry, a granulate or stabilized liquid, in a form suited for storage and shipping. Enzyme granulates may be made by known methods, e.g. by extrusion or drum granulation according to CA 974,907, US 4,106,991, US 4,435,307 or US 4,661 ,452. The stabilizer may be mixed with other ingredients in the core of the granules, preferably at above 1% by weight, preferably above 2%, particularly above 5%, and generally below 40%. Alternatively, the stabilizer may be incorporated by coating a preformed enzyme granulate. The amount of the stabilizer in the coating should make up at least 1% by weight of the coated particles. The coating may be applied by known methods e.g. as described in British Patent No. 1 ,362,365, page 1 , line 82 to page 2, line 34, and British Patent application No. 34973/73 and 10842/74 corresponding to Belgium patent No. 146,802. The coating material may comprise, in addition to the ethoxylated compound, other waxy coating materials and particulate material, e.g. magnesium silicate, alumino silicates, CaSO_4I CaCO₃, bentonite, zeolite or TiO₂.

A liquid enzyme preparation according to the invention may be stabilized against microbial infection by incorporation of a stabilizing agent known in the art. Examples are inorganic salts (such as NaCI), sugars (such as sucrose and glucose), polyols (such as glycerol, propylene glycol and sorbitol) and alcohols

(such as ethanol and iso-propanol). These are generally effective in amounts above

10%, especially above 20%. Another example is organic acids (such as benzoic, sorbic, propionic, lactic and formic), which are generally effective in amounts of 0.01- 2% at low pH (below 5). Other examples of stabilizing agents are antioxidants (such as sulphur dioxide), 1 ,2-benz-iso-thiazolin-3-one (BIT) and parabens. The amount of the dispersant in the liquid enzyme preparation is preferably 1-10% by weight.

An enzyme slurry may be produced according to WO 91/09941.

Determination of peroxidase activity (PODU) Peroxidase activity is determined by an assay based on the oxidation of 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate) (ABTS^®) by hydrogen peroxide (as described by Bergmeyer H.U., Methods of enzymatic analysis, 3rd edition, vol III, p. 286-293, 1983, modified). The greenish-blue colour produced is photometered at 418 nm. The analytical conditions are 0.88 mM hydrogen peroxide, 1.67 mM ABTS, 0.1 M phosphate buffer, pH 7.0, 30°C incubated for 3 minutes.

1 peroxidase unit (PODU) is the amount of enzyme that catalyzes the conversion of 1 xmol hydrogen peroxide per minute at these conditions.

Determination of catalase activity (CIU)

1 catalase unit (CIU) will decompose 1 μmole of H2O2 per minute at pH 7.0, 25°C, while the H₂O₂ concentration decreases from 10.3 to 9.2 μmoles per ml reaction mixture. The degradation of hydrogen peroxide is followed spectrophoto- metrically at 240 nm. EXAMPLES

EXAMPLE 1

Stabilization of peroxidase

Solutions were made in 0.1 M phosphate buffer at pH 7.0 containing 0.14, 0.28, 1.4 or 2.8 PODU/ml of Coprinus peroxidase and 1 g/l of stabilizer. The stabilizers tested were polyethylene glycol (molecular weight 6,000) and octylphenol polyethoxylate (mean degree of ethoxylation: 10; Triton X 405). The solutions were kept for 3 hours at 22°C and the peroxidase activity was determined before and after this incubation. Blank experiments were made in the same way but without stabilizer. The results shown in Table 1 are expressed as residual activity after 3 hours in % of the initial activity:

Table 1

The results show fairly rapid deactivation of peroxidase in the reference without stabilizer and full stabilization by the addition of 1 g/l of each stabilizer. Further tests showed full stabilization by use of 0.2 g/l of octylphenol ethoxylate at otherwise same conditions.

EXAMPLE 2

Stabilization of peroxidases Solutions were prepared in the same buffer as in Example 1 , containing horseradish peroxidase or Coprinus peroxidase, with or without octylphenol ethoxylate, and the peroxidase activity was measured by the ABTS^® method. Results:

The results show that the activity of both peroxidases is increased by about 10% by the addition of octylphenol ethoxylate.

EXAMPLE 3

Stabilization of peroxidase

Solutions of 0.2 ml about 0.1 PODU/ml of Coprinus peroxidase in 2 ml 2mM ABTS^® (pH 7.0) containing 0.2 ml 10.6mM H₂O₂ were incubated at 30°C. To some of the peroxidase samples had been added Triton X 405 (0.2 g/l), while others (controls) did not contain any stabilizer. In an additional experiment, 50 mM ammonium sulphate was added to the ABTS reagent before incubation. The results are shown in Table 2. It appears from Table 2 that an increase in enzyme activity of approx. 11% is obtained by addition of Triton X 405. The presence of ammonium sulphate results in an additional increase in registered OD of about 10%.

EXAMPLE 4

Dosage of stabilizer

The concentration of Triton X 405 in the peroxidase samples was varied from 0 to 1.5 g/l before incubation under the conditions described in Example 2, except that the temperature was 0°C. The enzyme activity was determined at 0, 1.5 and 3.5 hour. The results are shown in Table 3. It appears from Table 3 that an improved peroxidase stability is obtained by addition of 1 or 1.5 g/l of Triton X 405 to the 0.2 ml peroxidase sample (corresponding to a concentration of Triton X 405 of about 0.1 g/l during incubation).

Table 2

All figures are in KPODU/g

Table 3

All these figures are in KPODU/g

EXAMPLE 5

Enzvme dosage

The concentration of Coprinus peroxidase in the samples was varied from 0.3 - 0.6 PODU/ml to 3 - 6 PODU/ml. The concentration of Triton X 405 in the peroxidase sample was 1.5 g/l. Incubation with ABTS^® was carried out as described in Example 2, except that the temperature was 22°C. The enzyme activity was determined at 0, 1 , 2, and 3 hours. The results are shown in Table 4.

It appears from Table 4 that the presence of Triton X 405 stabilizes the peroxidase activity for up to 3 hours at both the lower and the higher peroxidase concentrations.

Table 4

All figures are in KPODU/g

The figures in parentheses indicate the residual activity in %

EXAMPLE 6

Stabilization of catalase

A catalase solution was obtained from a culture broth filtrate prepared according to WO 92/17571 by adding 15% propylene glycol and adjusting to pH 7.0. Stabilizer was added as shown in Table 5. Each solution was stored at 25 or 37°C for 2 or 4 weeks, the catalase activity was determined before and after storage, and results were expressed as residual activity in % of the initial activity. The same stabilizers were used as in Example 1.

Table 5

It appears from Table 5 that there is very little loss of catalase activity, even after 4 weeks of incubation.

Claims

1. A method of stabilizing a hemoprotein in an aqueous solution, characterized by incorporating a stabilizer of the formula:

R-O-(CHR'-CH₂-O-)_nH (I) wherein R = H or 0,-0^ alkyl, aryl or alkylaryl

R' = H or CH₃₎ and n = 4-50000.

2. A method according to the preceding claim of stabilizing an oxidoreductase, particularly a peroxidase (EC 1.11.1.7), an iodoperoxidase (EC 1.11.1.8) or a chloroperoxidase (EC 1.11.1.10), or a catalase.

3. A method according to the preceding claim, wherein the amount of said stabilizer corresponds to 0.2 - 5 mg per PODU (peroxidase activity).

4. A method according to any preceding claim, wherein the weight ratio of said stabilizer to the hemoprotein is in the range 1 - 50.

5. A method according to any preceding claim, wherein:

R is a C₁₀-C₁₈ linear or branched alkyl or alkylphenyl group and n is 5-50 (preferably 5-20).

6. A method according to any preceding claim, wherein:

R is H, and n is 50-500 (preferably 100-300).

7. A method according to any preceding claim, wherein the amount of said stabilizer is in the range 0.1 - 5 g per I of the aqueous solution.

8. A process comprising addition of peroxidase and of hydrogen peroxide or a precursor thereof, characterized by incorporating a stabilizer of the formula:

R-0-(CHR'-CH₂-0-)_nH (I) wherein R = H or 0,-0^ alkyl, aryl or alkylaryl R' = H or CH₃, and n = 4-50000.

9. A process according to Claim 8, further characterized as in any of Claims 3 - 6 or in an aqueous solution characterized as in Claim 7.

10. A process according to Claim 12 or 13 for bleaching of pulp for paper production, treating waste water from pulp production, or for lignin modification.

11. A peroxidase preparation in the form of a slurry, a granulate or a stabilized liquid, characterized by comprising a stabilizer of the formula:

R-O-(CHR'-CH₂-O-)_nH (I) wherein R = H or C^C^ alkyl, aryl or alkylaryl R' = H or CH₃, and n = 4-50000.

12. A peroxidase preparation according to Claim 11 , further characterized as in any of Claims 3 - 6.

13. A peroxidase preparation according to Claim 11 or 12, comprising 1-10% by weight of said stabilizer.

14. A coated granulate according to any of Claims 11 - 13, containing said stabilizer in the coating.