GB2218099A - Process for preparing a catalase-free oxidase - Google Patents

Abstract

The oxidase, eg methanol oxidase, is produced by aerobic fermentation of a catalase-negative yeast in the presence of: (a) a compound which induces the expression of the oxidase gene but is not a substrate for the oxidase, such as formiate or formaldehyde; (b) a so-called inducing substrate that induces expression of the oxidase gene and may also be substrate for the oxidase such as methanol; and (c) another source of carbon that is suitable for the yeast species chosen, the molar ratio of inducing compound not being a substrate : inducing substrate : other source of carbon being such that the yeast formed and the oxidase formed do not suffer any harmful effects from the oxidation of the inducing substrate. The ratios of (a), (b) and (c) are within the ranges given in EP-A-O 242 007 and EP-A-O 244 920, but the dilution rate has to be adapted to a relatively low value for optimal production of the oxidase, when the amount of (c) is relatively low compared with that of (a) and (b). For example, methanol oxidase is formed by induction with technical formaldehyde (a), containing some methanol (b), in the presence of glucose (c). Exemplified is the yeast Hansenula polymorpha, mutant 55/11, ATCC 46059. The catalase-free oxidase can be used for producing hydrogen peroxide, e.g. in a washing or bleaching process or for determination of ethanol or amines, or as a catalyst for oxidation reactions.

Description

PROCESS FOR PREPARING A CATALASE-FREE OXIDASE AND A CATALASE-FREE OXIDASE-CONTAINING YEAST, AND USE THEREOF FIELD OF INVENTION The invention relates to a process for the preparation of a catalase-free oxidase by aerobic fermentation of a yeast under conditions in which the yeast produces oxidase, and, if desired, isolation from the yeast cells of the catalase-free oxidase produced Oxidases are enzymes which catalyse specific oxidation reactions of certain substrates and thereby produce H202. An example of an oxidase is methanol oxidase (MOX) that enables methylotrophic yeasts such as Hansenula polymorpha, Candida boidinii, Pichia pastoris and suchlike to grow on methanol. The MOX catalyses the oxidation of methanol to formaldehyde and hydrogen peroxide during which molecular oxygen acts as electron acceptor.The formaldehyde formed is used for dissimilation (catabolism) during which it is converted into formic acid and finally into carbon dioxide, or used for assimilation (anabolism) during which cell material is produced via the xylulose monophosphate route. With these further processes other enzymes play an important role: in the assimilation process dihydroxyacetone synthetase is important in the dissimilation process formaldehyde dehydrogenase and formiate dehydrogenase are key enzymes.The hydrogen peroxide formed by the oxidase is extremely toxic for these organisms and, according to the present state of art, is immediately rendered innocuous by another enzyme namely catalase Other examples of oxidases are other alcohol oxidases glucose oxidase, glycerol oxidase D-aminoacid oxidase, aldehyde oxidase, amine oxidase, arylalcohol oxidase, galactose oxidase, sorbose oxidase, uric acid oxidase and xanthine oxidase.

Oxidases can be used for various purposes. An important use lies in the field of washing and bleaching compositions, where the presence of oxidase during the washing or bleaching process can lead to the in situ formation of hydrogen peroxide if at the same time a suitable substrate for the oxidase is present. The hydrogen peroxide displays a bleaching agent activity, or can, in co-operation with bleaching agent precursors such as TAED (etraacetylethylenediamine), lead to the formation of bleaching agents which are active at low temperatures, such as peracids.An important advantage of this system, in which the washing or bleaching agent itself contains no hydrogen peroxide but only the enzyme that during the washing or bleaching process catalyses the formation of hydrogen peroxide in situ, is that inactivation of washing composition components, such as proteases1 lipases and suchlike, during storage and transport is prevented. This use of oxidases is described in the British patent publications 1 225 713 and 2 101 167 and in the German patent publication 2 557 623.

Another use of oxidases lies in the field of qualitative and/or quantitative analysis, e.g. for determining the alcohol content of systems which are to be investigated, such as fermentation liquids. The analysis is based on a measurement of the hydrogen peroxide formed via an oxidation of colouring substances catalysed by peroxidase.

Oxidases can also be used within the framework of chemical syntheses or of processes for the purification of refuse in order to catalyse oxidation of substrates.

For the most of these uses, and certainly for that as component of washing or bleaching compositions and for that as aid in analysis, it is of great importance that the oxidase is free of catalase, which would decompose the hydrogen peroxide formed. A well-known problem with the microbiologically produced oxidases such as methanol oxidase (EC 1.1.3.13) by fermentation of methylotrophic yeasts such as Hansenula polymorpha, Candida boidinii, Pichia pastoris and suchlike on methanol, is, however, that they are constantly accompanied by the natural catalase enzyme tveenhuis M., van Dijken, J.P. and Harder, W. (1983) in Advances in Microbial Physiology, Rose, A.H., Gareth Morris, J.

and Tempest, D.W., Editors, Vol. 24, pp 1-82, Academic Press, New York] that almost immediately decomposes the peroxide formed, so that no effective bleaching agent activity or accurate analysis of the substrate concentration is possible. It is true that methods are known (see for example the British patent specification 2 101 167) for removing the catalase from the product to a great extent or for inactivating it and thus obtaining an active substantially catalase-free oxidase, but for technical application these methods (such as catalase removal through ion exchange chromatography or gel filtration, and catalase inactivation through treatment with sodium dodecylsulphate (SDS) or sodium azide, and so on) are far too expensive and cumbersome and in many cases inadequate, or, in the case of sodium azide, a toxic material is even introduced.

Consequently there is need of a method with which catalase-free oxidase can be obtained in a technically and economically justifiable manner. A microbiological production, in which catalase-negative mutants are used, could meet this need, but seems impracticable because, on the basis of the literature, it could be expected that the organisms without catalase are doomed to die if cultured in a nutritive medium to which substrate for the oxidase has been added in behalf of a good growth of the microorganism and strong expression of the oxidase gene.

BACKGROUND OF THE INVENTION In EP-A-O 244 920 (UNILEVER N.V. et al.) a process is described in which a microbiological production of methanol oxidase that is free of catalase can be realized on a technical scale with a process of the kind mentioned above, when a catalase-negative mutant of the yeast Hansenula polymorpha is allowed to grow in a nutritive medium suitable for the yeast in the presence of at least one compound which induces the expression of the oxidase gene but is not a substrate for the oxidase.

The scope of EP-A-O 244 920, however, is not limited to the production of methanol oxidase. With the aid of genetic engineering techniques, yeasts, particularly Hansenula and Pichia, can be obtained which contain another oxidase gene, preferably under control of the strong MOX promoter(s). Depending on the intended use, a need of such other oxidase, such as D-aminoacid oxidase and such-like, can exist.

In addition,instead of the catalase-negative Hansenula polymorpha indicated above, other catalase-negative yeasts can also be used. Depending on the oxidase to be produced, a compound other than formaldehyde or formiate might also be used as a compound which induces the expression of the oxidase gene, but is not a substrate for the oxidase. For example, according to K.B. Zwart and W. Harder, J. Gen. Microbiol. 129 (1983) 3157-3169, the formation of amine oxidase is induced in a medium containing 0.38 (w/v) glycerol and 5 mM ammonium sulphate, i.e. under ammonium-limiting conditions (cf. Table 2 on page 3161). Consequently, the scope of EP-A-O 244 920 is not limited to the production of methanol oxidase.

Therefore, in a general sense, the scope of EP-A0 244 920 embraces a process for the preparation of an oxidase or an oxidase-containing mixture or preparation by aerobic fermentation of a yeast under conditions in which the yeast produces oxidase, and, if desired, isolation from the yeast cells of the oxidase produced, wherein a catalase-negative mutant of a yeast is allowed to grow in a nutritive medium suitable for the yeast in the presence of at least one compound which induces the expression of the oxidase gene but is not a substrate for the oxidase.

In EP-A-O 242 007 (UNILEVER N.V. et al.) a very similar process is described in which a catalase-negative mutant of the yeast Hansenula polymorpha is allowed to grow in a nutritive medium suitable for the yeast in the presence of methanol and another source of carbon such as glucose, in which methanol induces the expression of the oxidase gene and can also be substrate for the oxidase beside the other source of carbon, while the toxic effects of the peroxide formed are prevented by using a suitable mixing ratio of methanol to other source of carbon.

Instead of the catalase-negative Hansenula polymorpha indicated above, other catalase-negative yeasts can also be used. Depending on the oxidase to be produced, a compound other than methanol should also be used as induction agent, that can also be substrate, for example methyl amine in the preparation of amine oxidase. Consequently, the scope of EP-A-O 242 007 is not limited to the production of methanol oxidase.

Various sources of carbon can be used instead of glucose. Therefore, in a general sense the scope of EP A-O 242 007 embraces a process for the preparation of an oxidase-containing composition by aerobic fermentation of a yeast under conditions in which the yeast produces oxidase, and, if desired, isolation from the yeast cells of the oxidase produced, wherein a catalase-negative mutant of a yeast is allowed to grow in a nutritive medium suitable for the yeast in the presence of: a) a so-called inducing substrate that induces expression of the oxidase gene and may also be substrate for the oxidase, and b) another source of carbon that is suitable for the yeast species chosen, the molar ratio of inducing substrate to other source of carbon being such that the yeast formed and the oxidase formed do not suffer any harmful effects from the oxidation of the inducing substrate.

In this description, "composition" means both a simple mixture of various components and a preparation in which the oxidase is packed, as it were, for example in encapsulated form.

DESCRIPTION OF THE INVENTION The present invention is based on experiments with the production of MOX by the catalase-negative mutant Hansenula polymorpha 55/11, ATCC 46059, described in the above-mentioned European patent applications, induced by technical formaldehyde.

On using a technical aqueous formaldehyde solution containing 37 wt.% formaldehyde and 13 wt.% methanol, it appeared that at a dilution rate of about 0.1 hours the MOX production was much less than would be expected on the basis of the experiments described in EP-A-O 244 920 (pure formaldehyde of pro analysi quality and glucose) and EP-A-O 242 007 (methanol and glucose). This result was disappointing and not expected, since, on the basis of the results described for pure formaldehyde and pure methanol, one might have expected that at the optimal dilution rate of 0.1 hours the common contribution of methanol and formaldehyde in the mixture (together 1.4 mole per 1 mole glucose) would give a similar high MOX production.

This disappointing result could be overcome by adapting the growth conditions. It was found that a good MOX production could be restored when the dilution rate was lowered to about 0.05 hour~l, which is surprising in view of the data given in Figure 2 of EP-A-O 244 920 showing that the MOX activity of MOX produced by Hansenula polymorpha 55/11 grown at D = 0.05 h~l is only slightly better than that at D = 0.1 h 1.

Based on this finding, the present invention comprises a process for the preparation of an oxidase or an oxidase-containing composition by aerobic fermentation of a yeast under conditions in which the yeast produces oxidase, and, if desired, isolation from the yeast cells of the oxidase produced, wherein a catalasenegative mutant of a yeast is allowed to grow in a nutritive medium suitable for the yeast in the presence of:: (a) at least one compound which induces the expression of the oxidase gene but is not a substrate of the oxidase, for example formaldehyde, (b) a so-called inducing substrate that induces expression of the oxidase gene and may also be substrate for the oxidase, for example methanol, and (c) another source of carbon that is suitable for the yeast species chosen, for example glucose, the molar ratio of inducing substrate to other source of carbon being such that the yeast formed and the oxidase formed do not suffer any harmful effects from the oxidation of the inducing substrate, whereby the dilution rate is adapted to the ratio of (a), (b) and (c) so that an appreciable amount of oxidase is formed.

As a combination of compounds (a) and (b), preferably technical formaldehyde is used.

It is preferred according to the invention that a catalase-negative mutant of a yeast of the genus Hansenula or of the genus Pichia, in particular of the species Hansenula polymorpha or of the species Pichia pastoris be used, such as the catalase-negative mutant Hansenula polymorpha 55/11, ATCC 46059. The invention is certainly not limited to the use of this one mutant.

Other catalase-negative mutants can be obtained by the introduction of any mutations at random and the subsequent selection of the mutants obtained, or by directed mutation methods, such as described inter alia in the European patent application 0 173 378 (A2).

It is further preferred according to the invention that a yeast be used in which the genetic information for the oxidase stands under control of a strong promoter1 as a result of which a large yield of oxidase can be realized. An example hereof is the MOX promoter, in particular the MOX promoter of Hansenula polymorpha CBS 4732. This occurs naturally in the Hansenula polymorpha 55/11, ATCC 46059 yeast described hereinbefore, but with other yeasts this can be achieved by means of known recombinant DNA techniques.

The strength of the promoter is apparent from the fact that the MOX content of the wild-type strain of Hansenula polymorpha cultured on methanol is in the order of magnitude of 30% of the cellular protein.

Examples of other strong promoters are the dihydroxyacetone synthetase (DAS) promoter of methylotrophic yeasts and the amine oxidase promoter of yeasts producing amine oxidase.

In connection with production on technical scale, it is preferred according to the invention that the yeast is cultured in a continuous fermentation. However, "batch" fermentation or "fed batch" fermentation is not excluded. By "fed batch" is meant a "batch" fermentation in which the substrate is not added in one go at the beginning, but gradually so as to achieve a controllable substrate concentration during the whole fermentation.

It is preferred, according to the invention, that, as oxidase gene, preferably a methanol oxidase gene is used, for example a methanol oxidase gene that codes for a methanol oxidase with the same amino acid sequence as the known MOX of Hansenula polymorpha CBS 4732, or a derivative of this sequence, obtained via enzyme engineering, or a modification thereof which hays the same functionality or a functionality that is better suited for substrates other than the well-known substrate for MOX. In combination with a methanol oxidase, preferably methanol is used as inducing substrate. As well as for methanol, the MOX enzyme also appears to be active for various other substrates, such as for ethanol, n-propanol, n-butanol, n-amylalcohol, and even, though to a slighter extent, for substances such as methylcellosolve, ethylene glycol, benzyl alcohol, isopropanol, isoamyl alcohol and propylene glycol. Some of these substances, such as ethanol, are pre-eminently suitable for serving as substrate when using methanol oxidase in washing and bleaching compositions, because they are relatively cheap and toxicologically acceptable.

During the fermentation, a nutritive medium suitable for the yeast, such as Hansenula or Pichia, is used which must contain a carbon source suitable for this organism. Suitable sources of carbon are known to the expert or can easily be determined by experiment.

Glucose, for example, is suitable, but also other sugars, such as sorbose, xylose, sorbitol, and other substances, such as glycerol and the like, can be used as carbon source, while also commercially available sources of carbon such as molasses come into consideration. A suitable nutritive medium is known from Egli et a1., Arch. Microbiol. 124 (1980) 115-121.

According to the invention it is important that, during the fermentation, a suitable ratio between the amount of compounds (a), (b) and (c) is established in combination with the dilution rate. Preferably, a relatively low dilution rate, e.g. about 0.05 h-l, is used.

The ratio of (a) to (c) and (b) to (c) is of the same order as described in the above-mentioned European patent specifications, thus preferably a molar ratio of formiate to carbon source of (0.5-4.5):1 or a molar ratio of formaldehyde to carbon source of (0.1-3):1 and a molar ratio of inducing substrate to other carbon source of (0.025-3):1.

According to a preferred embodiment of the invention, the catalase-negative oxidase-containing yeast is, after finishing the continuous fermentation, subjected to a drying treatment, whereby the oxidase enzyme is not inactivated, or another treatment whereby the cells are made permeable or the H202-decomposing system is inactivated (cf. EP-A-O 242 007). A freeze-drying treatment appeared to be very suitable.

The advantage of such drying treatment is that the cells with the H202-decomposing system inactivated by the drying treatment immediately start H202 production when used with methanol as the alcohol to be oxidised by the oxidase (cf. EP-A- 0 242 007).

If, for the intended use, one is unwilling or unable to use the cultured yeast cells as such, the oxidase accumulated in the yeast cells and particularly in the peroxisomes should be isolated from the cells. To do this, usual methods of cell lysis can be applied, such as the physical breaking of the cells with the aid of glass globules, treatment in a so-called French press or Manton Gaulin homogenizer, ultrasonic treatment, and enzymatic methods, e.g. with zymolyase.

The invention also relates to an oxidase-containing, catalase-negative mutant of a yeast, obtained by making use of a process according to the invention for preparing such a yeast, as well as to an oxidase or an oxidase-containing composition, obtained by isolation of the oxidase from a yeast according to the invention, possibly followed by processing the oxidase thus isolated to a composition with other components.

Further, the invention relates to the use of an oxidase-containing, catalase-negative mutant of a yeast according to the invention or the use of an oxidase isolated therefrom together with a substrate for the oxidase for the in situ production of hydrogen peroxide in a washing or bleaching process. Particularly when an alcohol oxidase is used, ethanol is preferably used as substrate in such a washing process.

Also a washing or bleaching agent that contains an oxidase-containing, catalase-negative mutant of a yeast according to the invention, or the oxidase isolated therefrom, is an embodiment of the invention.

Further, the invention relates to the use of an oxidase-containing, catalase-negative mutant of a yeast according to the invention or of an oxidase isolated therefrom as catalyst for the oxidation of a substrate for the oxidase within the framework of a chemical synthesis or of a process for purifying refuse or for the qualitative and/or quantitative determination of a substrate for the oxidase.

The invention is illustrated with the following Example.

Example I Species: Hansenula polymorpha, catalase-negative mutant 55/11, ATTC 46059.

Medium : see Table A.

Inoculum: a pre-culture was made in 200 ml Yeast Extract Peptone Dextrose medium for 24 hours at 300C.

Starting procedure: the fermenter, containing 9 1 basis medium with 40 g/l glucose, was inoculated and after one day continued with as continuous culture. The medium entering contained 88 g/l glucose.

Fermentation conditions: A 10 1 fermenter, with the trade name Chemoferm, having a working volume of 9 1 was used. The liquid level was adjusted by an exhaust pipe above the liquid. The pH was adjusted at pH 5.0 with 12.5% ammonia. Temperature 37"C. Stream of air 2 to 10 1/mien. The oxygen pressure was kept above 25% air saturation. Number of revolutions of the stirrer 750-1250 r.p.m. Foam formation was controlled by an automatic anti-foam regulation with Rhodorsil 426 R (Rhone Poulenc) as anti-foaming agent.

Analyses: all analyses were carried out as described in EP-A-O 242 007 and EP-A-O 244 920.

Induction of MOX in continuous cultures grown on technically pure formaldehyde/glucose After a steady state had been obtained on glucose at a dilution rate of 0.050 h-l, technically pure formaldehyde was put into the feeder stream. The feeder stream contained 88 g/l glucose and 42 g/l formaldehyde solution, containing 37% formaldehyde and 13% methanol.

In this way a molar ratio of formaldehyde : methanol glucose of 1.06 : 0.349 : 1 was obtained, which corresponds to a weight ratio of 15.5 : 5.5 : 88. After 118 hours a steady state of MOX activity of 3.9 Units/mg protein was obtained (see Table B). After the dilution rate had been increased to 0.078 h1, the MOX activity in the culture and the cell-free extracts decreased to very low levels. Lowering the dilution rate to 0.055 h'l resulted in a high MOX activity on both the culture and the cell-free extracts.

These results differ considerably from the results found in previous experiments described in EP-A0 244 920, in which pure formaldehyde in a formaldehyde/glucose mixture was used having a molar ratio of 1.8 mol formaldehyde : 1 mol glucose at a high dilution rate of 0.1 h-l, whereby 6 to 8 MOX units per mg protein was found. The addition of methanol to such mixture apparently lowers the MOX activity at high dilution rates. This in spite of the fact that, on the basis of the results described in EP-A-O 244 920 and EP-A-O 242 007, it was to be expected that, with the combined contribution of methanol and formaldehyde in the mixture (1.4 mol Eformaldehyde + methanol] : 1 mol glucose) at a dilution rate of 0.1 h'l, the MOX activity would likewise be high. Apparently the very low MOX activity at relatively high dilution rates could be increased to acceptable levels by decreasing the dilution rate to about 0.05 h-l.

It is not clear why the MOX activity is so low at higher dilution rates, since the concentrations of glucose, formaldehyde and formiate in the continuous culture are too low to be considered as toxic at both higher and lower dilution rates.

D glucose formaldehyde formiate h-1 g-1- g-1 g.1-1 0.050 < 0.02 #0.026 #0.01 0.078 < 0.02 $ 0.02 #0.01 0.055 < 0.01 < 0.04 0.01 The above data show that the remaining concentrations are negligible compared with the feedstock concentrations of glucose (88 g.l 1), formaldehyde (15.5 g.l'1) and methanol (5.5 g.l-1).

Production of hydrogen peroxide The cells cultured at a dilution rate of 0.055 h-l yielded, after freeze-drying, as described in EP-A0 242 007 and EP-A-O 244 920, a high hydrogen peroxide production capacity of 0.5 units per milligram dry biomass. This activity could be increased by permeabilization of the cells. Post-incubation for 30 to 40 minutes at 370C with a detergent such as Arquat R (0.05%) yielded an increase in the production of hydrogen peroxide to 0.7 units per milligram dry biomass.

In all cases, an absolutely catalase-free MOX preparation was obtained.

Table A - Composition of growth media g.1-1 Glucose 40 or 88 (see Example) Technically pure formaldehyde 0 or 42 (see Example) NH4C1 7.63 KH2PO4 2.81 MgS04. 7H20 0.59 CaC12.2H2O 0.055 FeSO4. 7H20 0.0375 MnSO4. H20 0.014 ZnSO4.7H20 0.022 CuSO4.5H2O 0.004 CoCl2.6H20 0.0045 Na2MoO4. 2H20 0.0026 H3B03 0.004 KJ 0.0026 EDTA 0.45 Biotine 0.000075 Thiamine HCl 0.00625 Meso-inositol 0.06 Pyridoxine 0.0015 D-pantothenic acid 0.03 Table B - Continuous culture experiment with technically pure formaldehyde Dilution rate Dry weight MOX activity MOX activity D (h-l) g/kg medium Units/mg in vivo protein Units/g d.w.

0.050 32.0 3.9 1.3 0.055 33.6 6.66 2.26 0.078 33.9 0.77 0.3

Claims (16)

1. CLAIMS 1. Process for the preparation of an oxidase or an oxidase-containing composition by aerobic fermentation of a yeast under conditions in which the yeast produces oxidase, and, if desired, isolation from the yeast cells of the oxidase produced, wherein a catalase-negative mutant of a yeast is allowed to grow in a nutritive medium suitable for the yeast in the presence of:: (a) at least one compound which induces the expression of the oxidase gene but is not a substrate for the oxidase, (b) a so-called inducing substrate that induces expression of the oxidase gene and may also be substrate for the oxidase, and (c) another source of carbon that is suitable for the yeast species chosen, the molar ratio of inducing substrate to other source of carbon being such that the yeast formed and the oxidase formed do not suffer any harmful effects from the oxidation of the inducing substrate, whereby the dilution rate is adapted to the ratio of (a), (b) and (c) so that an appreciable amount of oxidase is formed.
2. 2. Process according to claim 1, in which a catalase-negative mutant of a yeast of the genus Hansenula, preferably of the species Hansenula polymorpha, particularly of Hansenula polymorpha 55/11, ATCC 46059, or of the genus Pichia, preferably of the species Pichia pastoris is used.
3. 3. Process according to any one of the claims 12, in which formiate or formaldehyde is used as the compound (a) which induces expression of the oxidase gene but is not a substrate for the oxidase.
4. 4. Process according to claim 3, in which the molar ratio formaldehyde : carbon source such as glucose is 0.1:1 to 3:1.
5. 5. Process according to one or more of claims 14, in which technical formaldehyde is used as combination of (a) and (b).
6. 6. Process according to one or more of the preceding claims, in which, as oxidase, a methanol oxidase is produced and preferably that methanol is used as inducing substrate (b).
7. 7. Process according to claim 6, in which a methanol oxidase is produced that has the same amino acid sequence as the known methanol oxidase of Hansenula polymorpha CBS 4732, or a derivative of this sequence, obtained via enzyme engineering.
8. 8. Process according to one or more of the preceding claims, in which the yeast is cultured in a nutritive medium that contains another source of carbon (c), chosen from the group consisting of sugars, such as glucose, sorbose, xylose, sorbitol, commercially available carbon sources such as molasses, and other carbon sources used in microbiology, such as glycerol and suchlike.
9. 9. Process according to claim 8, in which methanol and glucose are used as sources of carbon (b) and (c), respectively,in a molar ratio of methanol glucose of (0.025-3):1, preferably (l-1.8):1.
10. 10. Process according to one or more of the preceding claims, in which the yeast is grown in a continuous fermentation.
11. 11. Process according to one or more of the preceding claims, in which a yeast is used that is modified with the aid of recombinant DNA techniques or its progeny, in which the genetic information for the oxidase stands under control of a strong promoter, preferably a methanol oxidase (MOX) promoter, particularly that of the MOX of Hansenula polymorpha CBS 4732.
12. 12. Process according to one or more of the preceding claims, in which the catalase-negative oxidase-containing yeast is, after finishing the fermentation subjected to a drying treatment, whereby the oxidase enzyme is not inactivated, e.g. a freezedrying treatment, or another treatment whereby the cells are made permeable or are inactivated.
13. 13. Oxidase-containing, catalase-negative mutant of a yeast, obtained by making use of a process according to one or more of the preceding claims.
14. 14. Oxidase or an oxidase-containing composition, obtained by isolation of the oxidase from the yeast according to claim 13, optionally followed by processing the oxidase thus isolated to a composition with other components.
15. 15. Use of an oxidase-containing, catalasenegative mutant of a yeast according to claim 13 or of an oxidase according to claim 14, together with a substrate for the oxidase, preferably ethanol, for (1) the in situ production of hydrogen peroxide in a washing or bleaching process, (2) the qualitative and/or quantitative determination of a substrate for the oxidase, or (3) the oxidation of a substrate of the oxidase within the framework of a chemical synthesis or of a process for purifying refuse.
16. 16. Washing or bleaching composition, characterized in that it contains an oxidasecontaining, catalase-negative mutant of a yeast according to claim 13 or of an oxidase according to claim 14.