AS AN ACTIVE COMPONENT

TECHNICAL FIELD

This invention relates to a hemopeptide with peroxidase activity, to a peroxidase preparation, and to production and use of such a preparation.

BACKGROUND ART

Peroxidase activity catalyses oxidation of a substrate (an electron or hydrogen donor such as lignin) with hydrogen peroxide.

High-molecular peroxidases (E.C. 1.11.1.7) are produced intracellularly by some microorganisms. Thus, S. Loprasert et al., Journal of General Microbiol- ogy (1988), 134, 1971-1976) describe a peroxidase-catalase from Bacillus stearo- thermophilus with molecular weight 175,000, and US 4,698,306 describes a per¬ oxidase from Coprinus with molecular weight 37,000-41 ,000. Also, so-called micro- peroxidases of mammalian origin are known; these are hemopeptides, typically with molecular weight in the range 1 ,500-3,000, exhibiting peroxidase activity (e.g. DE 3134526).

Use of peroxidase together with hydrogen peroxide or a hydrogen peroxide precursor has been suggested e.g. in bleaching of pulp for paper production (SE 88/0673), in treatment of waste water from pulp production (US 4,623,465, JP-A 2-31887) and for improved bleaching in laundry detergents (WO 89/09813). Pending US patent applicε n S.N. 07/421,414 (filed 13 October 1989) and a co-pending PCT application αisclose the use for dye transfer inhibition during laundering.

It is the object of the invention to provide an improved peroxidase for these purposes. The peroxidase should be active and stable at high temperature and H ₂ 0 ₂ concentration, especially at alkaline conditions. It should be free of

catalase as this activity breaks down hydrogen peroxide that is needed in the reaction. For better production economy, the peroxidase should be microbial and should be produced extracellularly by the microorganism in question.

STATEMENT OF THE INVENTION

5 We have, surprisingly, discovered that peroxidase is produced extracellularly by some strains of Bacillus pumilus. The novel peroxidase preparation from B. pumilus is a microperoxidase preparation, i.e. it contains hemopeptide as an active component. The preparation has improved stability at high temperature, at high pH and at high concentrations of hydrogen peroxide. It 10 can be produced without undesired catalase activity.

Accordingly, the invention provides a hemopeptide characterized by the general formula:

R - Cys-lle-Ser-Cys-His - R'

\ /

15 Heme

wherein the sulphur atoms of the two cysteine are linked to the vinyl groups of heme, and R and R' each represents a peptide chain of 0-10 amino acids.

The invention also provides a peroxidase preparation, characterized by comprising as an active component a hemopeptide as defined above or one

20 that contains a heme group linked to a peptide chain containing the sequence Gln- Glu-Gln-Thr-Xaa-lle-Ser-Yaa-His-Gly-Asp-Asn-Met-Gln, wherein Xaa and Yaa represent any amino acid. In another embodiment the invention provides a peroxidase preparation, characterized by comprising one or more active components derived from β. pumilus with molecular weight in the range 800-

25 2500, said preparation being further characterized by a pH optimum in the range

7.5-9, by retaining at least 50% residual activity after 2 hours at 80" C, and by having optimum activity at H ₂ 0 ₂ concentration above 20 mM.

The invention further provides a method of producing a peroxidase preparation, characterized by comprising cultivation of a peroxidase-producing strain of B. pumilus followed by recovery of peroxidase.

Finally, ^" the invention provides use of the above peroxidase prepara¬ tion together with hydrogen peroxide (optionally formed in situ) in bleaching of lignin-containing material.

DETAILED DESCRIPTION OF THE INVENTION

Hemopeptide

As noted above, one embodiment of the invention provides a microperoxidase, i.e. a hemopeptide with the general formula:

R - Cys-lle-Ser-Cys-His - R' \ / Heme

wherein the sulphur atoms of the two cysteine are linked to the two vinyl groups of heme, and R and R' each represents a peptide chain of 0-10 amino acids. These compounds can be prepared synthetically by methods known in the art, viz. by first synthesizing the peptide chain including the two cysteines and then letting this peptide react with heme, or they can be produced genetically by methods known in the art: a degenerate oligonucleotide probe is synthesized (based on the known amino acid sequence) and used to isolate the B. pumilus gene encoding the precursor peptide chain of the microperoxidase. Modified microperoxidases are hereafter obtained by b.te directed mutagenesis (ref. Molecular Cloning, A Laboratory Manual, Cold Spring Harbor, 1989, Ed. J. Sambrook, E.F. Fritsh and T. Maniatis, ISBN 0-87969-309-6).

In preferred embodiments, R is Gln-Glu-Gln-Thr- and R' is -Gly-Asp- Asn-Met-Gln; either of these may be shortened by one or more amino acids. This hemopeptide can be prepared by cultivation of a B. pumilus strain as described below. The following shows a comparison of the sequences of hemopeptides of the invention with a known microperoxidase (DE 3134526) prepared by hydrolysis of cytochrome C from animal or plant:

Prior art:

Phe-Val-Gln-Lys-Cys-Ala-Gln-Cys-His-Thr-Val-Glu-Lys-Gly \ /

Heme

Invention (preferred embodiment):

Gln-Glu-Gin-Thr-Cys-lle-Ser-Cys-His-Gly-Asp-Asn-Met-Gln

\ / Heme

It is seen that the novel microbial microperoxidase has very little homology with the known microperoxidase.

Cultivation of B. pumilus

B. pumilus strains that can be used in the practice of the invention are atypical in their ability to produce peroxidase. Some B. pumilus strains, including the type strain, have been found not to produce peroxidase.

Two peroxidase-producing strains are freely available with deposit numbers ATCC 12905 and NCIB 8600, and one strain (internal designation S 197) has been deposited by the applicant under the terms of the Budapest Treaty; the deposit date was 23 July 1990, and the deposit number is DSM 6124.

ATCC indicates the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, U.S.A., NCIB indicates the National Collection of

Industrial and Marine Bacteria Ltd., Torry Research Station, P.O. Box 31, 135 Abbey Road, Aberdeen AB9 8DG, Scotland, and DSM indicates Deutsche Sammlung von Mikroorganismen, Mascheroder Weg 1 B, 3300 Braunschweig, West Germany. Peroxidase-producing B. pumilus strains can be cultivated under aerobic conditions in a nutrient medium containing assimilable carbon and nitrogen together with other essential nutrient. The medium can be composed in accordance with principles known in the art.

During cultivation, the cells secrete peroxidase extracellularly, while they apparently produce catalase intracellularly. Extracellula" production of the peroxidase is advantageous as it allows for high fermentation yield and simple recovery. Catalase is undesired for most applications of peroxidase, so the recovery of peroxidase preferably includes separation of cell mass from the cell broth while avoiding cell lysis, e.g. by filtration or centrifugation. The resulting cell-free culture broth contains a mixture of hemopeptides with peroxidase activity, mainly with molecular weight in the range 800-2500. This can be used as such, optionally after concentration e.g. by evaporation or ultrafiltration. If desired, the various hemopeptides can be separated and purified to the desired degree by conventional methods, e.g. by column chromatography.

PH and temperature dependence

A crude peroxidase preparation of the invention (fig. 1) and a similar purified preparation (fig. 2) have slightly different pH optimum, but both are in the range 7-9. The thermostability of peroxidase preparations of the invention (fig. 3 and 4) is significantly better than a prior-art microbial peroxidase (fig. 5). A crude preparation according to the invention (fig. 3) is more thermostable than a similar purified preparation. Both preparations of the invention retain more than 40% residual activity after 30 minutes incubation at 80* C at pH 7.

Hvdroαen peroxide dependence

A peroxidase preparation of the invention (fig. 4) shows increasing reaction rate up to 20 mM H ₂ 0 ₂ (the highest tested). For comparison, a prior-art microbial peroxidase shows decreasing reaction rate at H ₂ 0 ₂ concentrations above 0.1 mM (fig. 7).

Bleaching of liαnin-containinα material

Due to its good stability at high temperature and high H ₂ 0 ₂ concentration, the peroxidase preparation of the invention is well suited for bleaching of lignin-containing materials, such as pulp for paper production or waste water from pulp manufacturing, together with hydrogen peroxide, e.g. as described in SE 88/0673, US 4,623,465 or JP-A 2-31887. Typical conditions are pH 7-10, 25-70° C, 10-300 mM H ₂ 0 ₂ and a dosage of 10-100 NOPA/g dry matter. Hydrogen peroxide may be added as such or formed in situ, e.g. by incorporation of a precursor, such as a perborate or percarbonate, or of an enzymatic system capable of generating hydrogen peroxide, e.g. an oxidase and a substrate therefor (such as glucose and glucose oxidase).

Other uses of peroxidase

Use of the peroxidase preparation of the invention is particularly advantageous at high temperature and/or high H ₂ 0 ₂ concentration and at alkaline pH. As an example, it can be incorporated into a laundry detergent together with a hydrogen peroxide precursor (such as sodium perborate or percarbonate) to improve the bleaching of stains according to WO 89/09813.

As another example, the peroxidase preparation can be used to inhibit the transfer of a textile dye from a dyed fabric to another fabric when the fabrics are washed and/or rinsed together in a wash liquor, by adding it together with a hydrogen peroxide precursor to the wash liquor in which the fabrics are washed and/or rinsed.

Peroxidase assay (NOPA)

Peroxidase activity is measured at 2 mM H ₂ 0 ₂ . The following are mixed in a 30° C thermostated 1 ml quartz cuvette:

200 μl 1 mM 4-aminoantipyrine (4-AA, Sigma No. A-4382, 0.2 mg/ml) 200 μl N-ethyl-N-sulphobutyl-m-toluidine-Na (ESBT, 5.86 mg/ml)

200 μl 0.5 M phosphate buffer, pH 7.0 200 μl enzyme sample, diluted to 0.02-0.10 NOPA/ml

200 μl 10 mM hydrogen peroxide is added, and the absorbance at

550 nm is followed for 1 minute. The activity is expressed in units denoted NOPA, calculated as the increase in absorbance within the first minute after addition of

H ₂ 0 ₂ multiplied by the dilution. The enzyme sample should be diluted so that the increase in absorbance per minute is within the limits 0.02 to 0.10.

BRIEF DESCRIPTION OF DRAWINGS

Figures 1 and 2 show the pH-activity curve of a crude and a purified peroxidase preparation, respectively, according to the invention, measured by the NOPA method, but varying the pH as shown.

Figures 3 and 4 illustrate the thermostability of a crude and a purified peroxidase of the invention, respectively. For comparison, figure 5 shows the thermostability of a prior-art peroxidase preparation (from Coprinus). The measurements were made by incubating at the indicated time and temperature, then immediately diluting a sample 10 times in 0.1 M phosphate buffer pH 7 at 25" C, and measuring peroxidase activity (NOPA).

Figures 6 ?nd 7 illustrate reaction at various H ₂ 0 ₂ concentrations with peroxidase of the invention and prior-art peroxidase (Coprinus), respectively. Oxidation of ESBT + 4-AA was followed by measuring the absorbance at 550 nm.

Figures 1 and 3 were made using cell-free culture broth prepared as in Example 1. Figures 2, 4, 6 and 7 were made using pool II from Example 1.

Figures 8 - 11 show chromatograms from purification of peroxidase of the invention. Details are given in Example 2.

EXAMPLES

EXAMPLE 1

Production of peroxidase from Bacillus pumilus

Media were prepared as follows (ingredients in g/l):

The medium was autoclaved at 121 ° C for 45 minutes

The agar was autoclaved at 121 ^β C for 45 minutes

Inoculum aoar : 10 Agar3 slants were inoculated with 1 freeze dried Bacillus pumilus Strain no S197 and incubated at 30 °C for 24 hrs.

Inoculum media : Two 500 ml Shake flasks containing 100 ml TY*3 media were inoculated with one Agar3 slant and incubated at 30" C and 250 rpm for 24 hrs.

Peroxidase production : 50 Shake flasks containing 100 ml of TY*3 were inoculated each with 2 ml of inoculum described above. Then 2.5 ml of a sterile 40% (w/w) glucose in water was added to each shake flask. The shake flasks were incubated at 30 °C for 48 hrs and then harvested. The final peroxidase activity was 1 NOPA/ml (NOPA measured at 20 mM H ₂ 0 ₂ ).

EXAMPLE 2

Purification of peroxidase from B. pumilus

3250 ml of the culture broth obtained in Example 1 was filtered through a Seitz Supra 100 filter plate and secondly through a Supra 50 plate to obtain a clear filtrate with an activity of 1.29 NOPA/ml.

To 900 ml of the filtrate was added 70 g Amberlite XAD16 hydro- phobic resin and the mixture was stirred overnight at 4°C. The resin was isolated by decantation and washed with 10%v/v ethanol. Peroxidase was extracted from the resin with 200 ml 50%v/v ethanol which was evaporated to 72 mi with an activity of 13.8 NOPA/ml (yield: 86%).

The evaporated perox -..ase extract was applied to a DEAE Sepharose F ^~ column (5x8 cm) equilibrated with 20 mM phosphate pH 7. The column was washed with more than one volume of buffer containing 0.1 M NaCI and peroxidase was eluted with buffer containing 0.3 M NaCI. Fractions of 10 ml were

collected and pooled in three pools according to protein (A280) and activity profiles (fig. 8):

A 90 ml sample with 7.34 NOPA/ml of pool C was added 1.6 M ammonium sulphate to a conductivity of 108 mS (pH 7.05) and applied on an Octyl-Sepharose column (5x11 cm) equilibrated with 0.8 M ammonium sulphate pH 7. Unbound material was eluted with 0.8 M ammonium sulphate and peroxidase activity was then eluted with a linear gradient of ammonium sulphate from 0.8 to 0 molar (pure water).

Fractions of 10 ml were collected and pooled in three pools according to the peroxidase activity (fig 9):

Fraction no. mi A280 A398 NOPA ml Yield

Pool I 84-92 90 0.26 0.19 1.47 20.0%

Pool II 94-96 30 0.58 0.37 2.09 9.5%

Pool III 97-100 40 0.55 0.30 1.57 9.5%

Total yield: 39%

A sample of pool I was concentrated by ultrafiltration on a 50 ml Amicσn stirred cell with a DDS GS90PP membrane. A 7.5 ml concentrated sample with an activity of 131.2 NOPA/ml was applied to a Sephacryl-S200 column (2.5x85 cm) equilibrated with 12.5 mM phosphate buffer pH 7.0 and eluted with the same

buffer at a flow rate of 0.9 ml/min. Fractions of 9.8 mi were collected and pooled according to peroxidase activity and spectral properties (Rz-value: A398/A280) (fig. 10):

Yield 43.3% 6.7% 15.8%

The mass spectrum of Pool IA show one major peak at Mw 10 2214.0/2228.1 and two minor peaks at Mw 972.8 and 1768.1, respectively. The amino acid sequence of the major component was shown to be:

Gln-Glu-Gln-Thr-?-lle-Ser-?-His-Gly-Asp-Asn-Met-Gln

where the two missing amino acids both are believed to be cysteine through which a heme group (responsible for the A398 absorbance) can be bound.

15 A sample of pool II was concentrated by ultrafiltration on a 50 ml

Amicon stirred cell with a DDS GS90PP membrane. A 10 ml concentrated sample with an activity of 143.8 NOPA/ml was applied to a AcA 202 column (2.5x85 cm) equilibrated with 12.5 mM phosphate buffer pH 7.0 and eluted with the same buffer at a flow rate of 0.4 ml/min. Fractions of 5 ml were collected and monitored for

20 A280, A398 and peroxidase activity (fig. 11).

This AcA 202 gel is claimed by the manufacturer to separate molecules in the range from 1 kD to 15 kD. From the chromatogram it is seen that a wide range of relatively low Mw substance was eluted and all showing the same peroxidase activity relative to the A398 (Soret) absorbance.

EXAMPLE 3

Bleaching of kraft pulp

The peroxidase used in this example was the permeate from ultrafiltration of the supernatant from alcohol precipitation of a cell-free culture broth (obtained essentially as in Example 1).

1 g hardwood (birch) kraft pulp, delignified by oxygen, was blended in 250 ml water adjusted to pH 8.0-8.2 with NaOH. H ₂ 0 ₂ was added to a concentration of 1% (0.3M), and the above peroxidase preparation was added to a dosage of 0.32 NOPA/ml. The mixture was incubated at 40 °C for 2 hours. Then the pulp was collected on a Buchner funnel and washed with sufficient water to obtain a clear filtrate. The pulp was pressed and dried, and the ISO brightness of the obtained sheet was measured. A reference was made in the same way without the enzyme.

Invention: 57.2% ISO brightness Reference: 55.6% ISO brightness

EXAMPLE 4

Bleaching of CTMP pulp

The experiment of Example 3 was repeated at 30 "C with CTMP softwood (pine) pulp. Invention: 59.7% ISO brightness

Reference: 56.1% ISO brightness

EXAMPLE 5

Bleaching of dves in solution

The effect of the peroxidase preparation of the invention in bleaching a dissolved textile dye was tested. The dyestuffs tested were Direct Blue 1 (C.I. #24410, product of Keystone Aniline), Acid Red 151 (C.I. #26900, product of Sandoz), Procion Blue H ERD (product of ICI) and Procion Blue H EXL (product of ICI). The peroxidase preparation was cell-free culture broth prepared essentially as in Example 1.

A reaction solution was prepared, containing 50 mM sodium phosphate, 0.3 NOPA/ml of peroxidase, dyestuff (as indicated below) corresponding to an absorption maximum (in the visible range) of 0.025-0.035, and 0.25 mM H ₂ 0 ₂ at room temperature at the pH indicated below. After addition of H ₂ 0 ₂ (added last), a spectral scan was made every minute for 12 minutes. Below, the change in absorbance at the wavelength of maximum absorption is listed.

When the two values of the absorbance change are close, the bleaching is practically instantaneous. Generally, bleaching over the entire visible range follows the above trends at the absorbance maximum.

In all cases, use of 0.25 mM H ₂ 0 ₂ without enzyme left the dye unchanged.

This example illustrates the potential usefulness of the peroxidase preparation of the invention in preventing surplus dye from strongly-coloured fabric from being redeposited during washing.

Inttπurttontl Application No: PCT/ /

MICROORGANISMS

Optional Meet In connection wtth th* mtcroβrganlwn referred M on M«e 4 ~Z , in* = 7-=~. -J -— ^~ > o* t_M deeeripβen >

A. IOINTIΠCATION OP OIPOSIT •

Further deposit* ar* WwiMM on an addWonal eh**t Q»

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23 JULY 1990 DSM 612-4

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