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Patent 2360751 Summary

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(12) Patent: (11) CA 2360751
(54) English Title: MULTICOMPONENT SYSTEM FOR ENZYME-CATALYZED OXIDATION OF SUBSTRATES AND PROCESS FOR THE ENZYME-CATALYZED OXIDATION
(54) French Title: SYSTEME MULTICOMPOSANT POUR L'OXYDATION DE SUBSTRATS CATALYSEE PAR DES ENZYMES ET PROCEDE D'OXYDATION CATALYSEE PAR DES ENZYMES
Status: Deemed expired
Bibliographic Data
(51) International Patent Classification (IPC):
  • D21C 9/147 (2006.01)
(72) Inventors :
  • CANDUSSIO, ANTON (Germany)
(73) Owners :
  • CONSORTIUM FUR ELEKTROCHEMISCHE INDUSTRIE GMBH (Germany)
(71) Applicants :
  • CONSORTIUM FUR ELEKTROCHEMISCHE INDUSTRIE GMBH (Germany)
(74) Agent: OSLER, HOSKIN & HARCOURT LLP
(74) Associate agent:
(45) Issued: 2005-01-04
(86) PCT Filing Date: 2000-02-17
(87) Open to Public Inspection: 2000-09-08
Examination requested: 2001-07-10
Availability of licence: N/A
(25) Language of filing: English

Patent Cooperation Treaty (PCT): Yes
(86) PCT Filing Number: PCT/EP2000/001290
(87) International Publication Number: WO2000/052257
(85) National Entry: 2001-07-10

(30) Application Priority Data:
Application No. Country/Territory Date
199 09 546.9 Germany 1999-03-04

Abstracts

English Abstract



The invention relates to a multi-component system for carrying out mediator-
dependent enzymatic oxidation, comprising an oxidation
catalyst, an oxidant and a mediator and characterised in that a) the oxidation
catalyst is chosen from the group comprising manganese
oxidases; b) the oxidant is selected from the group comprising oxygen and
oxygen-containing compounds; c) the mediator is selected from
the group comprising compounds containing Mn ions.


French Abstract

L'invention concerne un système multicomposant pour réaliser une oxydation catalysée par des enzymes, en fonction du médiateur. Ce système qui comprend un catalyseur d'oxydation, un agent oxydant et un médiateur, est caractérisé en ce que a) le catalyseur d'oxydation est sélectionné dans le groupe des manganèse oxydases, b) l'agent oxydant est sélectionné dans le groupe comprenant l'oxygène et les composés contenant de l'oxygène, c) le médiateur est sélectionné dans le groupe des composés contenant des ions Mn.

Claims

Note: Claims are shown in the official language in which they were submitted.



THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A multicomponent system for mediator-dependent
enzymatic oxidation comprising an oxidation catalyst, an
oxidizing agent, and a mediator, characterized in that
a) the oxidation catalyst is selected from the group of
manganese oxidases,
b) the oxidizing agent is selected from the group
consisting of oxygen and oxygen compounds,
c) the mediator is selected from the group of compounds
which contain Mn ions.
2. The multicomponent system as claimed in claim 1,
characterized in that it additionally comprises a complexing
agent selected from the group of complexing agents that can
complex Mn ions.
3. The multicomponent system as claimed in claim 1 or
2, characterized in that the mediator is Mn2+ or Mn3+ ions.
4. The multicomponent system as claimed in claim 2,
characterized in that the complexing agent does not
contain nitrogen, is readily biodegradable and is
toxicologically harmless.
-41-


5. The multicomponent system as claimed in one of
claims 1 to 4, characterized in that the oxygen is present
either directly in the gaseous form or in the form of liquid
oxygen or as oxygen-containing gas mixture.
6. A process for oxidizing a substrate, characterized
in that a manganese oxidase, in the presence of oxygen and,
if appropriate, a complexing agent for Mn ions, forms Mn3+ by
direct Mn2+ oxidation, and in that the Mn3+ ion oxidizes the
substrate, itself being reduced to Mn2+ and, in turn, being
available for direct oxidation by the manganese oxidase.
7. The process as claimed in claim 6, characterized in
that the substrate is used in the form of an aqueous
solution, mixture or suspension.
8. The process as claimed in claim 6 or 7,
characterized in that manganese ions are used at
concentrations between 0.005 mM and 50 mM.
9. The process as claimed in claim 6, 7 or 8,
characterized in that oxygen having a partial pressure of
0.05 - 5 bar is used.
10. The process as claimed in one of claims 6 to 9,
characterized in that the complexing agents are used at
concentrations between 1 mM and 500 mM.
-42-

Description

Note: Descriptions are shown in the official language in which they were submitted.




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Multicomponent system for enzyme-catalyzed oxidation of
substrates and process for the enzyme-catalyzed oxidation
The present invention relates to a multicomponent system
for mediator-dependent enzyme-catalyzed oxidation of
substrates and a process for the enzyme-catalyzed oxidation.
Multicomponent systems for mediator-dependent enzyme-
catalyzed oxidation of substrates and corresponding processes
are known in principle. Mediators in this context are
generally those compounds which can be oxidized by
oxidoreductases, that is firstly also substrates of enzymatic
oxidation catalysts: Mediators are particularly distinguished
by the oxidized form of the mediator (the activated mediator,
for example a mediator free radical or a mediator cation)
having a sufficiently long life in order to diffuse from the
oxidoreductase to the~actual substrate of the oxidation
system and to interact with this. In the interaction between
activated mediator and substrate the substrate is oxidized by
the mediator. By the oxidation of the substrate the mediator
can either be regenerated (catalytic oxidation system) or
inactivated (stoichiometric oxidation system). If the
mediator is regenerated, it is available for a new catalysis
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cycle. The electrons transferred from the substrate to the
oxidoreductase by the mediator are transferred to terminal
electron acceptors such as oxygen or peroxide in direct
enzymatic oxidation processes.
In the case of processes where peroxide serves (directly
or released from precursors) as terminal electron acceptor,
only peroxidases can be used as oxidoreductases. Although a
number of such processes are known, they are burdened with
numerous disadvantages:
Peroxidases are too expensive in manufacture in order to
be used in industrial processes such as paper bleaching or in
laundry detergents. In addition, the addition of peroxidases
is a problem, since, in the concentrations necessary for the
efficacy of the process, peroxide inactivates the peroxidase.
Many of the compounds described as mediators for peroxidases
.cannot be used industrially because they are too intensively
colored, are toxicologically or ecotoxicologically harmful,
are not biodegradable or only poorly biodegradable and are
not sufficiently available in sufficient amounts or cheap
enough. Processes in which peroxide serves as terminal
-2-



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electron acceptor have therefore not been suitable to date
for economic expedient industrial use.
In the case of processes in which oxygem acts as
terminal electron acceptor for the electrons originating from
the substrate to be oxidized, oxidases are used as
oxidoreductases. Two groups of oxidases are described, which
can be used in mediator-dependent enzymatic oxidation
processes: laccases (Boubonnais & Paice (1990) FEBS LETTERS
Vol 267 (1), p. 99 - 102, W0 94/29510) and tyrosinases (~WO
94/29510). In contrast to the case with peroxidases, in which
a prosthetic heme group acts as redox-active center, the
electron transfer in the case of laccases and tyrosinases is
catalyzed by four copper ions coordinating with four
corresponding copper-binding domains of these oxidases ('blue
copper' oxidases). A total of four electrons are sequentially
taken up from mediators which are then transferred in a four-
electron transfer to molecular oxygen. The oxygen is reduced
to water, and the oxidase is regenerated for a new reaction
cycle. A number of mediators which catalyze the electron
transfer from the substrate to be oxidized to the oxidases
-3-



,
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are described, for example, in WO 94/29510, WO 97/06244, WO
96/10079 or WO 95/01426.
It is known to use these mediator-dependent enzymatic
oxidation processes involving oxidases and oxygen for coal
liquefaction (WO 94/29510), for wood pulp bleaching (WO
94/29510), for organic synthesis (Potthast et al. (1995) J.
Org. Chem., Vol. 60, p. 4320 - 4321), as a bleaching system
in laundry detergents and dishwashing detergents (WO
97/06244), for denim bleaching (WO 96/12846), for preventing
dye transfer during washing (WO 98/23716) or for breaking
down polycyclic aromatic hydrocarbons (Johannes et al. (1996)
Appl. Microbiol. Biotechnol., Vol. 46,. p. 313 - 317).
However, since the mediators have properties which stand
in the way of industrial use, the enzymatic oxidation
processes which use oxidases also have serious disadvantages:
- The mediator HBT which is described as particularly
effective in WO 94/29510 is not biodegradable (Amann (1997)
9th Int. Symp. on Wood and Pulp. Chem., F4-1 - F4-5).
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- Many mediators such as HBT and violuric acid in their
active form inactivate the oxidases used (Amann (1997) 9th
Int. Symp. on Wood and Pulp. Chem., F4-1 - F4-5) and
therefore require high enzyme usage in the respective
process.
- A group of very active mediators is characterized by
an N-0 group and thus contains at least one N atom per
mediator molecule (WO 94/29510). From this there result the
problems known with the use of N-0-containing compounds with
regard to toxicity and biodegradability. Biological
wastewater purification systems in the papermaking pulp
industry can, in addition, frequently not process additional
N loads.
- Some of the mediators, for example ABTS, form
intensively colored free-radicals and therefore lead to an
unwanted discoloration of the substrate to be oxidized.
- Most of the mediators contain N or S atoms and are
therefore relatively expensive to manufacture.
_5_



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These disadvantages have also limited to date wide
industrial use of such systems for oxidizing substrates.
Archibald & Roy (Archibald & Roy (1992) Appl. Environ.
Microbiol. Vol. 58, p. 1496 - 1499) described a process in
which a redox cascade consisting of phenols and Mn2'/Mn3+, acts
as mediator between oxidase and the substrate to be oxidized.
As terminal electron acceptor, the system uses oxygen. The
oxidase used is a laccase. The primary substrate oxidized by
the laccase is initially phenols such as m-hydroxybenzoi-c
acid or m-cresol. In the presence of complexing agents such
as pyrophosphate ions, secondary oxidation of Mnz' to Mn'' can
then take place with the participation of the oxidized
phenols. For this system, it could not be demonstrated that
nonphenolic substrates could also be oxidized. Also, with a
system in which only Iaccase, Mn2+ and a complexing agent was
used for lignin oxidation, delignification could not be
found. This mediator-dependent enzymatic oxidation system.
does not represent technically any significant improvement
compared with the other mentioned systems, because, instead
of the mediators, it uses a combination of phenols, .
complexing agents and Mn ions and the obligatory use of
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phenolic mediators hinders the use of the process for
toxicological reasons. In addition, the long reaction times
of > 15 h hinder practical use of this system.
The object of the present invention was to provide a
multicomponent system for a mediator-dependent enzymatic.
oxidation of substrates which does not have the disadvantages
of the known multicomponent systems and therefore makes
possible simple and inexpensive conversion of the substrate
to be oxidized.
The object is achieved by a multicomponent system
comprising an oxidation catalyst, an oxidizing agent, and a
mediator, characterized in that
a) the oxidation catalyst is selected from the group of
manganese oxidases,
b) the oxidizing agent is selected from the group
consisting of oxygen and oxygen compounds,



CA 02360751 2001-07-10
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c) the mediator is selected from the group of compounds
which contain Mn ions.
Preferably, the inventive multicomponent~system
comprises a complexing agent which is selected from the group
of complexing agents that can complex Mn ions.
For the purposes of the present invention, manganese
oxidases are to be understood as those oxidases which oxidize
Mn or Mn ions directly and which transfer the resulting
electrons to oxygen. Preferably, such oxidases are able to
oxidize Mn'+ to Mn'+ in the presence of oxygen and complexing
agents. Preference is given to manganese oxidases which
contain copper ions as the redox-active catalytic group.
Manganese oxidases can be isolated, for example, from
known microorganisms. For this, such microorganisms are grown
under conditions under which they form manganese oxidases.~In
the inventive composition, the manganese oxidases used can be
in the simplest case enzymatically or chemically disrupted
cell preparations of the complete manganese oxidase-
containing microorganisms. However, it is also possible to
_g_



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use manganese oxidase-containing culture supernatants or
isolated manganese oxidases.
Manganese oxidases are formed, for example, by
Leptothrix discophora, Bacillus SG-1 and Pseudomonas sp.
(Nealson et al. (1989) Beveridge & Doyle (eds.) Metal ions
and bacteria, John Wiley and sons, Inc., New York, pp. 383-
411). The genes coding for manganese oxidases from Leptothrix
discophora and Bacillus SG-1 are cloned and sequenced
(Corstjens et al. (1997) Geomicrobiol. Journal, Vol. 14,' p.
91-108 and van Waasbergen et al. (1996) Journal of
Bacteriology, Vol. 178 (12), p. 3517 - 3530). Both genes code
for proteins which contain the copper-binding sequences known
from 'blue copper' oxidases and their manganese oxidase
activity is increased by adding copper.
In addition to said manganese-oxidase-producing
microorganisms, other microorganisms can be used as a source
of manganese oxidases. Thus, for example, in the case of
microorganisms where the manganese oxidase is accumulated in
the spore, for example Bacillus SG-1, the isolated spores can
also be used in the inventive process as enzyme source.
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CA 02360751 2001-07-10
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In addition, manganese oxidases can be used which are
produced by recombinant production processes. Recombinant
production processes are taken to mean in this case all
processes in which the genes coding for manganese oxidases
are isolated from the natural producers and have then been
introduced by known methods into suitable production strains,
which can also be the original enzyme producers.
Manganese ions act as mediators in the inventive
composition. These manganese ions can be added to the process
in any oxidation state.
Preferably, manganese ions of oxidation state +2 or +3
are used.
Manganese of oxidation state +2 is preferably used in
the form of manganese sulfate or manganese chloride:
Manganese of oxidation state +3 is preferably used in
the form of soluble manganese complexes. Examples of such
manganese complexes are manganese/formate, manganese/.lactate,
manganese/oxalate or manganese/malonate.
-10-



a
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WO 00 ! 62257 PCTIEP0010Q1290
The mediator (for example the Mn3+ ion) takes up one
electron from the substance to be oxidized. This oxidizes the.
substance, and the Mn ion itself is reduced (for example to
Mn2+ ion). In this form the Mn ion transports~the electron
which is taken up to the manganese oxidase, releases it there
and is oxidized back to the initial oxidation state (for
example to the Mn3+ ion). Oxygen acts as oxidizing agent for
the manganese oxidase and thus as terminal electron acceptor.
The oxygen can be produced directly in the reaction by
means of known chemical or enzymatic oxygen generating
systems. It can also be produced by electrical hydrolysis of
water, or it can be used directly in the gaseous or liquid
state.
Preferably, oxygen is used either directly in the
gaseous state or in the form of liquid oxygen or as oxygen-
containing gas mixture, for example air. Particularly
preferably, oxygen is introduced as oxygen-containing gas
mixture, for example air.
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Preferably, the Mn3+, which is formed by direct
oxidation of Mn2+ by manganese oxidases, acts in the presence
of a complexing agent.
Complexing agents which are suitable are preferably
compounds which complex Mn3+ and thus stabilize it.
Preferably, complexing agents are used which do not
contain nitrogen, are readily biodegradable and are
toxicologically harmless. Such complexing agents are, far
example, formate, lactate, malonate or oxalate.
The inventive system has the following advantages
compared with known systems:
- The problem of inactivation of the oxidoreductases,
as occurs, for example, when peroxide is used as
electron acceptor, does not occur in the case of
the inventive composition, since oxygen acts as
oxidizing agent and thus as terminal electron
acceptor.
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- Oxygen can be added in a technically simple manner
and in sufficient amounts.
- Manganese oxidases, as is known, do not require
prosthetic groups, for example heme groups, for
their activity, in contrast to peroxidases and also
manganese peroxidases. Thus manganese oxidases_are
to be prepared in customary production systems on
an industrial scale without problems.
- The inventive system does not require N-containing
or S-containing mediators or colored or poorly
biodegradable or toxic mediators. The only redox-
active mediator acting is manganese ions, which
alternate between two oxidation states, preferably
the oxidation states 2+ and 3+. An inactivation of
this manganese mediator cannot take place by a
chemical modification, for example, in contrast to
the inactivation of known mediators.
The inventive composition may be used for oxidizing the
most varied substrates. Preferably, it may be used to oxidize
those substrates which can be oxidized by Mn ions.
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Particularly preferably, it may be used for oxidizing those
substrates which can be oxidized by Mn3+ ions.
The inventive multicomponent system is suitable, for
example, as a bleaching system in laundry detergents and
dishwashing detergents, for wood pulp bleaching, for denim
bleaching, for treating wastewater, for organic synthesis,
for preventing dye transfer during washing or for breaking
down polycyclic aromatic hydrocarbons. A further potential
use is, for example, coal liquefaction.
Corresponding processes are described in the prior art
for mediator-dependent enzymatic oxidation processes using
oxidases and oxygen.
It is easy for a~person skilled in the art to modify the
respectively known processes with use of the components and
process conditions specified in the present application and
to use the inventive composition for said purposes.
The present invention further relates to a process for
oxidizing a substrate, characterized in that a manganese
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WO 00 I 52257 PCTIEP0010G1290
oxidase, in the presence of oxygen and, if appropriate, a
complexing agent for Mn ions, forms Mn3+ by direct Mn2+
oxidation, the Mn3+ ion oxidizes the substrate, itself being
reduced to Mn2+ and, in turn, being available~for direct
oxidation by the manganese oxidase.
In the inventive process, the substrate is preferably
used in the form of an aqueous solution, mixture or
suspension.
In the inventive process, preferably, between 0.001 and
50 milligrams of active manganese oxidase per liter of
reaction volume are used.
The manganese oxidase is preferably introduced into the
reaction mixture in granular form, as a solution, as a
suspension or with a support material.
Particularly preferably, the manganese oxidase is
introduced into the reaction mixture in the form of a
suspension which comprises between 0.5 and 50 percent by
weight of enzyme in a nonionic detergent.
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In the inventive process, preferably, manganese of the
oxidation states Mn2+ or Mn3+ is used as redox mediator.
Manganese ions oz oxidation state 2+ or 3+ can be used
directly in this oxidation state, or can be produced in the
process from manganese ions of other oxidation states, such
as Mn4+ or Mn7+.
Manganese of oxidation state +3 is preferably added in
the form of soluble manganese complexes such as
manganese/formate, manganese/lactate, manganese/ oxalate or
manganese/malonate.
Preferably, in the inventive process, manganese of
oxidation state +2 is used. Particularly preferably,
manganese sulfate or manganese chloride is used.
In the inventive process, manganese ions are used at
concentrations between 0.005 mM and 50 mM. Preferably,
manganese ions are used at concentrations between 0.05 mM and
mM, and particularly preferably at concentrations between
0.1 mM and 1 mM.
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PCTIEPOOIOQ1290
Depending on the specific application, the required
manganese may already be present in the substrate to~be
oxidized. One example of an inventive process in which
generally no external manganese ions need to be added is wood
pulp. bleaching. Wood and the pulp obtained therefrom
frequently already naturally contain a sufficiently high
amount of manganese ions for the inventive oxidation process.
In the inventive process, oxygen is preferably used at a
partial pressure of 0.05 - 5 bar. Particularly preferably,
oxygen at a partial pressure zrom 0.1 to 2.5 bar is used. In
particular preferably, oxygen at a partial pressure of from
0.2 to 1 bar is used.
In the inventive process, said complexing agents are
preferably used at concentrations between 1 mM and 500
mM. Preferably, suitable complexing agents are used at
concentrations between 5 mM and 100 mM, and particularly .
preferably at concentrations between 10 mM and 50 mM.
The inventive oxidation process can be used for
oxidizing all compounds which can be oxidized by Mn3+ ions.
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WO 00 l 52257 PCTIEP0010Q1290
Inventive processes can be used, for example, for the
oxidation of lignin in papermaking and wood pulp production,
as oxidation system in laundry detergents and dishwashing
detergents, for enzymatic dye bleaching, for enzymatic
textile bleaching, as a system for preventing dye transfer
during washing, for the specific oxidation of organic target
molecules in organic synthesis, for oxidative wastewater
treatment, for degrading chlorinated hydrocarbons, for
cleaving polycyclic aromatic hydrocarbons a.nd for liquefying
coal.
The examples below serve for further illustration of the
invention.
Example 1:
Isolation of manganese oxidase-producing microorganisms
l.a. Isolation of manganese-oxidizing microorganisms:
Manganese-oxidizing microorganisms, particularly fungi
and bacteria, were isolated on indicator plates by the method
of Krumbein & Altmann (Krumbein & Altmann (1973) Helgolander
wiss. Meeresunters., Vol. 25, pp. 347 - 356).
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In the presence of manganese ions of oxidation states
Mn3+ - Mn7+, Berbelin blue forms an intensive blue dye at pH
4 - 7. At an elevated concentration of Berbelin blue, in the
presence of suitable Mn ions the blue oxidation product is
formed up to pH 10.
For the plate screening, colorless Berbelin blue (N, N'-
dimethylamino-p,p'-triphenylmethane-o " -sul.fonic acid) was
added to nutrient media. At concentrations of 10-4 - 10-S g
of Berbelin blue per 100 ml of nutrient media no inhibition
of bacterial growth occurred.
To isolate heterotrophic manganese oxidizing bacteria,
the following medium was used:
3.5 g/1 Difco-Bacto-Peptone
0 . 8 g/ 1 MnS04 ~ HZO
100 mg/1 FeS04 ~ 7 H20
15 g/1 Difco-Bacto-Agar
750 ml of seawater
245 ml of distilled water
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ml of Berbelin blue stock solution (4 g/1)
pH 7.6
Bacteria-containing samples such as seawater samples,
sediment samples, soil samples, soils from ore deposits,
etc., were applied to such indicator plates at a suitable
dilution or concentration such that, per indicator plate,
between 100 and 300 individual colonies grew.
Bacteria which, after 5 to 10 days of incubation on such
indicator plates, formed blue colonies (i.e. the manganese-
oxidizing activity was cell-associated) or had blue haloes
around the colonies (i.e. manganese-oxidizing enzymes were
secreted into the culture supernatant), were analyzed
further.
l.b. Detection of manganese oxidases in Berbelin blue-
oxidizing microorganisms
Microorganisms which were isolated as described in la
can produce manganese oxidases, but the blue coloration could
also have been caused by manganese peroxidases or by
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oxidation of Fe2+. The specific detection of manganese
oxidase activity was carried out as described by Boogerd & de .
Vrind (Boogerd & de Vrind (1987) J. Bacterioi., Vol. 169 (2),
pp. 489 - 494):
First, the microorganisms under test were each grown for
days in 1 1 of the medium used for isolation without agar
or Berbelin blue addition. The cells were then removed by
centrifugation (15 min, 1 000 x g, 4°C). In the case of cells
which formed blue colonies on the indicator plates described
in example la, the cell pellet was used for the gel
electrophoresis described below. In the case of cells which
formed blue haloes on indicator plates, the cell-free culture
supernatant was concentrated by ultrafiltration to one
fiftieth of its initial volume on a OF membrane having a
retention capacity for particles > 10 000 D. The concentrate
or the cell pellet was mixed with the same volume of a buffer
.(0.125 M Tris/C1 pH 6.8, 20~ glycerol, 2~~SDS, 10$ 2-
mercaptoethanol, 0.01 bromophenol blue). 50 ul aliquots of
such a mixture were electrophoretically separated on a 10~
polyacrylamide gel. After the electrophoresis the gel was
washed four times for 15 min with deionized water: The gel
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was then incubated for 2 h in 100 uM MnCl2 in 10 mM Hepes pH
7.5. In samples which contained the manganese oxidases,
during this procedure, at the point at which after the
electrophoresis the manganese oxidases were localized in the
gel, a brown precipitate of manganese oxide formed. The
manganese oxide formation thus observed was caused in each
case by manganese oxidases directly oxidizing Mn2+, because
no peroxide was present and no ions other than Mn2+ were
present in the mixture.
Example 2:
Production and isolation of manganese oxidases
2.a. Manganese oxidase-containing spores from Bacillus
By the method described in example 1, Bacillus isolates
were isolated which had incorporated manganese oxidase into
their spores. For industrial uses, such manganese oxidase-
containing spores were used directly or the manganese
oxidase-containing spore coats were prepared and used. Such
spores or spore coats were produced as follows:
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Growth:
To produce manganese oxidase-containing spores, a
suitable Bacillus strain was grown aerobically at 25°C in the
following medium:
2 g/1 of peptone (Difco), 0.5 g/1 of yeast extract
(Difco), 10 ug/1 of iron-EDTA, 100 ug/1 of sterile-filtered
MnCl2 x 4 H20 in 50 mM Tris in 80o natural seawater, pH 7Ø
After a growth time of 10 days, > 95o of all cells had
sporulated.
Spore preparation:
The spores were harvested by centrifugation (30 min
000 x g, 4°C), washed with deionized water and suspended
_in 10 mM Tris/C1 pH 7.0 (0.1 g/ml). For a preparation of the
spore coat, this material was further processed as described
below. If the spores were used directly in the inventive
process, these were further treated as follows:
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50 ug/ml of lysozyme were added to the suspension, the
mixture was incubated for 30 min at 37°C, then the spores
were washed with 1M NaCl, 0.15 M NaCi, O.Io SDS and five
times with deionized water. Spores purified in this manner
were stored at 4°C in deionized water until use in the
inventive process.
Preparation of the spore coats:
The entire procedure was carried out at room
temperature, unless stated otherwise. Except for the 1~ SDS
solution and the deionized water used for washing, all
solutions contained 10 mM EDTA, pH 7.5 and PMSF (0.3 percent
by weight). The purified spores were suspended as described
above in 10 mM Tris pH 7.0 (0.1 g/ml) and the same volume of
glass beads (diameter l0 - 50 um) was added. The suspension
was then treated for 15 min in 30 sec intervals at 0°C with
an ultrasonic cell disrupter (Sonifier) at maximum amplitude.
After the treatment the suspension was incubated on ice for 5
min; the supernatant was then taken off from the settling
glass beads. The glass beads were then washed twice with a
volume of 10 mM Tris pH 7.0 corresponding to their own
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CA 02360751 2001-07-10
WO 00 l 52257 PCTlEP00/0Q1280
volume. The first supernatant and the supernatants from the
washing procedure were combined and centrifuged for l5 min at .
15 000 g. The supernatant was then taken off, the precipitate
was suspended in 10 mM Tris pH 7.5 and treated for 30 min at
37°C with lysozyme (100 ug/ml). The mixture was again
centrifuged for 15 min at l5 000 g. The precipitate
containing the spore coats was washed with: 1 M NaCl, 0.14 M
NaCl, 1~ SDS and 5 times with deionized water.
2.b. Manganese oxidase from Leptothrix discophora
Sheath-forming organisms such as Leptothrix discophora -
isolates which produce a manganese oxidase, can be obtained
by the method described in example 1. Such isolates can also
be obtained from commercial strain collections (Leptothrix
discophora ATCC 51168, Leptothrix discophora ATCC 51169).
Those which are particularly suitable for producing~manganese
~oxidase are derivatives of Leptothrix discophora which have
lost the ability to form sheaths. Such derivatives arise
spontaneously when sheath-forming strains are cultured
continuously and over a relatively long time under laboratory
conditions (Adams & Ghiorse (1986) Arch. MicrobioT., Vol.
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CA 02360751 2001-07-10
WO 00 / 52257
PCTIEP0010Q1290
145, pp. 126 - 135). Such derivatives which no longer form
sheaths secrete manganese oxidases into the culture medium.
The example below describes the production and isolation
of manganese oxidases into, and out of, the culture
supernatant of derivatives.of Leptothrix discophora which can
no longer form sheaths and are deposited in accordance with
the Budapest Convention at the DSMZ Deutsche Sammlung von
Mikroorganismen and Zellkulturen GmbH, D-32.124 Brunswick)
under the number DSMZ 12667:
Culture:
Leptothrix discophora, to produce manganese oxidase, wa.s
cultured in a medium which contained the following
constituents:
0.25g/1 Difco-Peptone


0.25g/1 Difco yeast extract


0.25g/1 glucose


0. g/1 MgSO~ 7 H20
6


0.07g/1 CaCl2 2H20


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CA 02360751 2001-07-10
WO 00 ! 52257
PCTIEPOOIOQ1290
0.015 g/1 MgS04 ~ H20
per liter of deionized water.
Before autoclaving the medium, the pH was set to pH 7.6
with 1 M NaOH.
To produce manganese oxidase, 45 1 of the medium
described were inoculated with two liters of a Leptothrix
discophora preliminary culture in the same medium. Culture
was performed with aeration (0.2 volumes of air per unit
volume medium and minute) at 26°C for 40 hours.
Enzyme preparation from culture supernatant:
After 40 hours culture was ended. The cells of
Leptothrix discophora were separated from the culture
supernatant by centrifugation. The cell-free, manganese
oxidase-containing culture supernatant was concentrated to
0.5 1 by ultrafiltration on a OF membrane having a.retention
capacity for particles > 10 000 D. The concentrated culture
supernatant was used as manganese oxidase source for the
examples described below.
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CA 02360751 2001-07-10
WO 00 I 62257 PCTlEPOOIOQ1290
Example 3:
Quantitative determination of the activity of manganese
oxidases using N,N,N~,N~-tetramethyl-p-phenylenediamine
( TMPD )
Mn3+ ions are able to oxidize the colorless compound
TMPD. The oxidation product 'bluster blue' has an intensive
blue color, and the increase in this color can be followed
photometrically at a wavelength of 610 nm.
Procedure:
A TMPD stock solution of 2.1 mM TMPD in distilled water
was prepared freshly before each measurement. The enzyme
preparation to be assayed was diluted in 10 mM HEPES (N-2-
hydroxyethylpiperazine-N'-2-ethanesulfonic acid pH7.5) so
that in the assay of activity described below, an OD 6T0
between 0.5,and 1.5 was measured after 10 minutes of assay
time.
The manganese source used was a solution of 10 mM MnS04
in distilled water.
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CA 02360751 2001-07-10
WO 00 ! 52257 PCTIEP00/0Q1290
To determine the manganese oxidase activity in enzyme
samples, in parallel, 3 ml aliquots of the enzyme dilutions
in HEPES (see above) were mixed with 0.3 ml of TMPD stock
solution. To one of each of the parallel assay solutions, at
a time point 0 min, 10 ul of the MnS09 stock solution were
added. Both samples were mixed and incubated for 10 min at
40°C. After 10 min the samples were briefly centrifuged (15
sec; 5 000 rpm), then the OD610 was determined in 1 ml of
each of the MnSOq-containing supernatants. The reference used
was the comparison sample without MnSOQ. An increase in .
absorption at 610 nm by 1.0 is equivalent to the formation of
100 um of 'bluster blue'. One unit (lU) of manganese oxidase
was defined as the amount of enzyme which leads to the
formation of 1 ~zM of 'bluster blue' from TMPD in 1 min.
For the subsequent assay, in each case concentrated
manganese oxidase-containing culture supernatants from
example 2 b and 50 ul of the spore coat suspension from
example 2 a were used. The following results were obtained:
Enzyme from ~ OD610
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_ ~ CA 02360751 2001-07-10
WO 00 ! 52257 PCTIEP00/0Q1290
Leptothrix ( 1.03
discophora
Bacillus 0.85
Example 4:
Oxidation of the homogeneously available substrate veratryl
alcohol as a function of varying manganese concentrations and
complexing agents
Procedure:
A stock solution of 0.25 g/ml of the substrate veratryl
alcohol (3,4-dimethoxybenzyl alcohol (Aldrich)) in ethanol
was prepared.
The oxidation reaction was carried out at 45°C with
gentle stirring. For this MnS04 was added (0.00, 0.05, 0.50
mM MnS04) was added to 22.23 ml in each case of a solution
containing a complexing agent (50 mM formate, lactate, malate
or oxalate; in each case pH 7.5). Then, 0.268 ml of the
veratryl alcohol stock solution was added in each case. The
mixture was equilibrated at 45°C for l0 min, then the
reaction was started by adding 2.5 ml of enzyme solution
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CA 02360751 2001-07-10
WO 00 I 52267 PCTJEP0010(a1290
(manganese oxidase from Leptothrix discophora, 10 U/ml).
After 8 h the reaction was studied by HPLC analysis for the
formation of veratryl aldehyde. For this, 0.4 ml of an H2SOq
solution (0.5 mol/1) was added to 0.8 ml of assay solution:
20 ul of this sample were applied to a LiChrospher 60 RP
Select B separation column (Merck 50940) and eluted with a
mixture of H20/MeOH (65:35). The flow rate was 1 ml/min. The
products in the eluate were detected by a UV detector at 275
nm.
The table below shows in o the amount of veratryl
alcohol oxidized to veratryl aldehyde in 8 h under the
conditions described in the process of the invention:
Complexing MnS04 Without Mn With Mn oxidase
agent [mM] oxidase from L.
[$ v. aldehyde] discophora
[~ v. aldehyde]


50 mM formate 0 0.00 0.00


50 mM formate 0.05 0.01 7.24 w


50 mM formate 0.5 0.00 12.46


50 mM lactate 0 0.00 0.00


50 mM lactate 0.05 0.00 3.32


50 mM lactate 0.5 0.02 2.79


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~

CA 02360751 2001-07-10
WO 00 / 52257 PCTIEP00I0G1290
50 mM malonate 0 0.00 0.00


50 mM malonate 0.05 0.03 14.23


50 mM malonate 0.5 0.02 22.96


50 mM oxalate 0 0.00 0.00


50 mM oxalate 0.05 0.06 17.30


50 mM oxalate 0.5 0.07 21.44


Example 5:
Wood pulp bleaching
Oxygen-delignified softwood kraft pulp having a
lignin content of kappa 16.5 was washed and brought to a pulp
density of 5o with 30 mM Na oxalate buffer (pH 7.5). MgSOq
(0.5 mM) was then added. The suspension was homogenized for
60 sec using a suitable stirrer. Then, in two parallel
solutions, 5 U of manganese oxidase (Leptothrix, Bacillus)
were added in each case per g of pulp. A third solution
without enzyme served as control. The solutions were
homogenized again for 60 sec and introduced into a
controlled-temperature steel autoclave which permitted gas'
introduction. The autoclave was sealed, an oxygen pressure of
3 bar was applied and the autoclave was incubated at 45°C for
4 h. The reaction solution was then discharged. The pulp was
washed with 10 volumes of flowing water at 50°C, then lignin
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CA 02360751 2001-07-10
wo 0o r szzs~ Pcrr~POOroa~zao
fragments were extracted under alkaline conditions from the
pulp using an aqueous NaOH solution at 70°C. 20 g of NaOH
were used per kg of pulp for the extraction. After the
extraction the pulp was again washed with 10 volumes of
flowing water at 50°C. The lignin content of the pulp samples
was finally determined via.the kappa value according to
standardized test method SCAN=C 1:77 of the 'Scandinavian
Pulp and Paper Board':
Enzyme Kappa value Delignification in $


Control without enzyme 15.2 7.9


Leptothrix discophora 13.4 18.8


Bacillus spore coats 10.7 35.1


Compared with the control sample, when the
inventive oxidation process is used, an additional
delignification between 10.9 and 27.2 was achieved.
'Example 6:
Suppression of dye transfer
The experiments to demonstrate the suppression of dye
transfer by inventive oxidation systems were carried out in
-33-



. CA 02360751 2001-07-10
WO 00 I 52257 PCTIEP00l0Q1280
heatable glass beakers (100 ml in volume). The control used
was a solution in which 0.25 g of a commercial detergent had
been dissolved in 50 ml of water. The experiments with
manganese oxidase were carried out in 50 ml of a 30 mM Na
malonate buffer (pH 7.5) containing 0.5 mM MnSOq. The 50 ml
assay solutions were first heated to 45°C, then 0.5 U/ml of
manganese oxidase was added where appropriate iri each case to
the buffer solutions. The cotton textile samples (5 g in each
case) were added to the assay solutions, then 10 ml of a
solution of 15 uM Cibacron Marine C-B (Ciba), preheated to
45°C, were added as test dye to each assay solution. After
incubation for one hour at 45°C, the textile samples were
removed from the assay solutions and each washed with 1 1 of
flowing water at 50°C. The textile samples were then washed
and dried. The brightness of the initial samples and the
treated samples was then determined spectroscopically:
In the table the brightnesses of the treated cotton
samples is compared with an untreated sample:
Enzyme Malonate buffer Water/
/ MnCl2 detergent
-34-



CA 02360751 2001-07-10
WO 00 / 52257 PCTIEP00I0Q1290
none 73.3 89~


Leptothrix discophora 89.20


Bacillus 92~ _


The values shown in the table indicate that the
inventive oxidation system is suitable for preventing
transfer of the added dye to the cotton samples. The effect
is comparable in quality to the effect achievable with
commercial detergents.
Example 7:
Stain bleaching (laundry detergent application)
In order to demonstrate the stain-bleaching action of
the inventive process, washing experiments were carried out.
For this, standardized soiling was produced by applying
dropwise 0.3 ml of an_aqueous solution of 100 ppm of Evan's
blue (obtainable from wako Pure Chemical Industry Co., Osaka)
in each case to white cotton samples (7.5 cm x 7.5 cm). These
textile samples were then treated according to the invention
with manganese oxidase, MnS04 and suitable complexing agent
in the presence of oxygen in heatable glass beakers (100 ml
volume) with stirring (magnetic stirrer). The control used
-35-



CA 02360751 2001-07-10
wo 0o r saas~ Pcrr~POOroQ~aso
was assay solutions in which a) only malonate buffer was
used, or b) no MnSOq was added, or c) no manganese oxidase
was added:
The experiments were carried out in 50 ml of a 30 mM Na
malonate buffer (pH 7.5) with or without 0.5 mM MnS09. The 50
ml test solutions were first heated to 45°C, then 1 U/ml of
manganese oxidase from Bacillus or from Leptothrix was added
to each of the test solutions which were to contain manganese
oxidase. The soiled cotton samples were added to the test
solutions. After incubation for one hour at 45°C, the textile
samples were removed from the test solutions and washed in
each case with 1 1 of flowing water at 50°C. The textile
samples were then dried.
To determine the differences in color of the samples
compared with the control sample without manganese oxidase
and MnSOq, the Y, y and x values of the air-dried cotton
samples were measured using a color difference meter (CR-200,
Minolta). To assess the bleaching effects of the inventive
processes, the Z value (Z=(1-x-y)Y/y) was determined from the
resulting values.
-36-



CA 02360751 2001-07-10
WO 00 I 52257 PCTIEP0010Q1290
The table shows the whitenesses of the measured samples
compared with the whiteness of the control without enzyme and .
without ~InSOq
+ MnSOq - MnSOq


Bacillus 2.7 -0.3


Leptothrix 1.6 0.1


no enzyme 0.2 0.0


It can be seen from the table that the whiteness of
samples containing manganese oxidases and MnSOq was increased
by between 1.6 and 2.7 points compared with samples without
enzyme and without MnSOq.
Example 8:
Organic synthesis
The examples shown below demonstrate that an inventive
process is suitable for the targeted synthesis of organic
substances.
a) Oxidation of alcohol groups to aldehydes
269 mg (1.6 mmol) of.3,4-dimethoxybenzyl alcohol in 1 ml
of ethanol were added at 45°C to 22 ml of a 30 mM malonate
-37-



w " CA 02360751 2001-07-10
WO 00 f 52257 PCTlEP0010Q1290
solution pH 7.5 containing 0.5 mM MnSOq. After incubation for
min, the manganese oxidase (40 U) was added. After a
reaction time of 24 h, the reaction solution was extracted
with chloroform and studied by NMR spectroscopy. In the case
of manganese oxidase from Leptothrix, the yield was 32$ 3,4-
dimethoxybenzaldehyde, and,in the case of manganese oxidase
from Bacillus, the yield was 27~ 3,4-dimethoxybenzaldehyde.
b) Oxidation of alcohols to ketones
196 mg (1.6 mmol) of 1-phenylethanol were added at 45°C
to 22 ml of a 30 mM malonate solution pH 7.5 containing 0.5
MnS04. After incubation for l0 min, the manganese oxidase (40
U) was added. After a reaction time of 24 h, the reaction
solution was studied by HPLC. In the case of manganese
oxidase from Leptothrix, the yield was 17~ acetophenone, and
in the case of manganese oxidase from Bacillus, the yield was
19~ acetophenone.
In both examples, the inventive oxidation process was
successfully used for the synthesis of defined substances.
The substrates used are intended here only to demonstrate the
oxidation capacity of the process in an exemplary manner and
not to restrict the region of synthesizable products.'
-38-



~ r~ CA 02360751 2001-07-10
WO 00 ! 52267 PCTIEP0010Q1280
Example 9:
Denim bleaching
Pieces of dyed denim fabric cut into squares (9 g / 160
cm2) were incubated together with manganese oxidase, 0.2 mM
MnS09 and 15 mM complexing agent (oxalate) in a closed 500~m1
beaker in a total liquid volume of 11.5 ml at 45°C. The pH of
the test solutions was 7.0 (15 mM oxalate). 10 U of manganese
oxidase were added per gram of textile. After incubation for
4 hours, the textile pieces were washed under flowing water
until the wash water was colorless. The textile pieces were
dried in a sheet dryer, then pressed and optically assessed
using a suitable spectrophotometer.
The degree of bleaching was determined using a CM 3700d
spectrophotometer (Minolta) in accordance with the
manufacturer's instructions. Measurements were made without
gloss and without UV. The brightness of the samples~was
determined as the percentage of total reflection compared
with a whiteness standard (R 457). L* is the measure of
brightness (white = 100; black = 0).
-39-



r. CA 02360751 2001-07-10
WO 00 ! 52257 PCTIEP0010G1290
The values of the treated textiles were compared with
the values of untreated reference textiles. The change in
brightness (DL*) of the samples compared with untreated
controls was calculated using the software PP2000
(Opticontrol).
~L*


Control 0


Leptothrix 23.14


Bacillus 28.72


A change in brightness (DL*) of 5 is already visible to
the naked eye, that is to say significant bleaching action
was achieved with both manganese oxidases.
-4 0-

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Administrative Status

Title Date
Forecasted Issue Date 2005-01-04
(86) PCT Filing Date 2000-02-17
(87) PCT Publication Date 2000-09-08
(85) National Entry 2001-07-10
Examination Requested 2001-07-10
(45) Issued 2005-01-04
Deemed Expired 2007-02-19

Abandonment History

There is no abandonment history.

Payment History

Fee Type Anniversary Year Due Date Amount Paid Paid Date
Request for Examination $400.00 2001-07-10
Registration of a document - section 124 $100.00 2001-07-10
Application Fee $300.00 2001-07-10
Maintenance Fee - Application - New Act 2 2002-02-18 $100.00 2002-02-07
Maintenance Fee - Application - New Act 3 2003-02-17 $100.00 2003-02-13
Maintenance Fee - Application - New Act 4 2004-02-17 $100.00 2004-02-12
Final Fee $300.00 2004-10-20
Maintenance Fee - Patent - New Act 5 2005-02-17 $200.00 2005-02-02
Owners on Record

Note: Records showing the ownership history in alphabetical order.

Current Owners on Record
CONSORTIUM FUR ELEKTROCHEMISCHE INDUSTRIE GMBH
Past Owners on Record
CANDUSSIO, ANTON
Past Owners that do not appear in the "Owners on Record" listing will appear in other documentation within the application.
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Description 2001-07-10 40 1,160
Description 2001-07-11 40 1,164
Abstract 2001-07-10 1 54
Claims 2001-07-10 2 62
Claims 2001-07-11 2 62
Cover Page 2001-12-07 1 33
Claims 2004-05-20 2 60
Cover Page 2004-12-02 1 33
PCT 2001-07-10 11 414
Assignment 2001-07-10 3 150
Prosecution-Amendment 2001-07-10 9 245
Correspondence 2001-12-19 1 41
Assignment 2002-01-17 3 145
Correspondence 2002-02-28 1 19
Prosecution-Amendment 2002-08-21 1 54
Fees 2003-02-13 1 52
Correspondence 2004-10-20 1 45
Prosecution-Amendment 2003-12-15 2 46
Fees 2004-02-12 1 49
Prosecution-Amendment 2004-05-20 3 67
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