PROCEDE POUR L'OBTENTION D'ENZYMES LIGNINOLYTIQUES

PROCESS FOR OBTAINING LIGNINOLYTIC ENZYMES

Abrégé anglais

ABSTRACT



The extraction of ligninolytic enzymes by cultivation of white-
rot fungi can be improved in that at the point of incipient
colouration at least 0.1 g/l, in particular 0.3 to 5 g/l, and
especially 1 g/l of manganese dioxide powder, in particular
finely divided powder with a grain size of less than 10 µm, is
added to the fungal culture and the pH value in the medium is
kept constant at a value between 4.8 and 5.0 during the whole of
the culturing period. It is preferred that the enzymes be
periodically separated off from the culture medium by ultra-
filtration and that the residual culture substrate be recycled.

Revendications

PATENT CLAIMS

1. A process for obtaining ligninolytic enzymes by the
cultivation of white-rot fungi, characterized in that at the
start of colouration at least 0.1 g/l manganese dioxide
powder is added to the fungal culture and the pH value in
the medium was kept constant at between pH 4.8 and 5.0
during the total duration of the culture.



2. A process as defined in claim 1, characterized in that the
manganese dioxide is added in the form of finely divided
powder with a grain size of less than 10 µm.



3. A process as defined in claim 1 or claim 2, characterized in
that the manganese dioxide is added in quantities of 0.3 to
5 g/l and in particular of 1 g/l of culture medium.



4. A process as defined in one of the preceding claims,
characterized in that the manganese dioxide is added to a
culture with fungi cultivated on a carrier.



5. A process as defined in one of the preceding claims,
characterized in that the enzyme is periodically separated
off from the culture medium and the residual culture
substrate is recycled.




6. A process as defined in one of the preceding claims,
characterized in that a strain of Phanerochaete
chrysosporium is cultivated as the white-rot fungus.

Description

;~OC~490

The present invention relates to a process for obtaining
ligninolytic enzymes by the cultivation of white-rot fungi.



Ligninolytic enzymes, the so-called ligninases, are key enzymes
in the biological breakdown of lignin and lignocelluloses. For
this reason, they may in the future play a considerable role in
the wood and paper industry, in so-called bio-pulping, for
cleaning waste water, etc. For this reason, processes that
permit the production of large quantities of these enzymes are
thus of particularly great interest.



Thus, it is the aim of the present invention to describe a
process by means of which the production of such ligninases is
greatly enhanced.



The process developed according to the present invention for this
purpose is characterized in that at the beginning of colouration,
o.l g/l of manganese dioxide powder is added to the fungal
culture and the pH value within the medium is kept constant
between pH 4.8 and 5.0 for the whole duration of the culture.



Most surprisingly, it has been found that the addition of
manganese dioxide to the fungal culture leads to a considerable

increase of extractable ligninolytic enzymes. This seems to
ensure both an increased production of the enzymes as well as
their stabilization relative to the inactivating influences of




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the culture liquid: the hydrogen peroxide that i5 formed by the
fungi simultaneously with the enzvmes discussed herein, and
through which the enzymes can be rendered inactive, is broken
down catalytically by the addition of manganese dioxide to the
fungal culture, on the cell walls of which the MnO2 builds up so
that the fungal mycelium appears to be black after the addition.
The mycelium treated in this way can be used for ligninase
production over a longer time, in that the culture liquid is
separated off and recycled by ultra-filtration and optionally the
replenishment of the substrate once the ligninases have been
extracted.



The manganese dioxide is added in a finely divided form, in
particular with a grain size of less than 10 ~m, preferably in
quantities of from 0.3 to 5 g/l, and especially at about 1 g/l of
culture medium. Commercially available manganese (IV) oxide
powder (pulvarized pyrolusite with an average grain size of 7 ~m)
was used. However, any other finely divided form of manganese
dioxide (with the exception of dead-burned oxide) can be used in
the same manner.



Within the so-called agitation cultures, the fungi can be in the
form of mycelium balls or pellets, or cohesive mycelium mats, or
can be cultivated on carriers.




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All the strains of P. chrysosporium tested up to now have been
found to be suitable; in most instances, a two-fold increase in
enzyme production could be effected by the addition of the
manganese dioxide. According to the present invention, the
stabilizing effect of the manganese dioxide does not result in
the usually observed reduction of enzyme activity, so that the
economy of this ligninase extraction could be considerably
improved by this means.



The fungal mycelium treated with the manganese dioxide can be
stored for long periods at refrigerator temperatures (+4C). The
following example explains the present invention.



Example:
The following strains were cultivated: ATCC 32629, Novobranova;
ATCC 24725 and strain SC26, a mutant of the strain ATCC 24725.



After preculturi~g in a glucose medium (2 days at 39C in air, in
Fernbach flasks) the mycelium that was formed was washed with
distilled water and homogenized in distilled water under sterile
conditions. Aliquote portions of this homogenate were used as
the innoculum for the test batches. Based on proven and
publicized media (see T.K. Kirk et al, Enzyme Microb. Technol. 8
(1986) 27-32 and H.W. Kern et al, Appl. Environ. Microbiol. 53
(1987) 2242-46), a culture medium of the following composition (N

deficit) was used (per litre):




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K2HPO4, 4.59 mmol; KH2PO4, 8.8 mmol; MgSO4, 2 mmol; CaC12, 0.68
mmol; thiamine, 2.97 ~mol; ammonium tartrate, 1.1 mmol; trace
elements, 10 ml of a stock solution as in T.K. Kirk (see above);
veratryl alcohol 0.4 mmol; Tween 80 0.5 g.



Glucose or glycerine, both at 10 g/l, were used as a C-source.
The pH value of the culture liquid was adjusted by KOH. An
additional buffering of the medium during cultivation was
effected by the addition of cis, trans-aconite acid (6 mmol/l).



The fungi were cultivated in various sizes of Erlenmeyer flasks
(200 ml flasks with 70 ml or l-litre flasks with 300 ml of the
innoculated medium). After the flasks had been flushed with
oxygen they were closed with silicon stoppers and incubated at
39C with an agitation frequency of 130 rpm (Pilot-shaker,
Braun).



After approximately 2 days of incubation the fungal mycelia that
had grown into balls (so-called pellets) displayed an incipient
colouration (dark brown). At this time, solid and sterilized -
manganese dioxide (MnO2) was added to the cultures (l g
MnOJlitre) which was then flushed once again with oxygen, ;
whereupon cultivation was continued under the same conditions.




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The drastic difference in ligninase production by cultures,
effected with or without the addition of MnO2, can be seen from
the following table:



Duration of culture 3 d 4 d 5 d
_________________________________________________________________

Ligninase in U/l* without MnO2 301 570 410

with MnO2 309 1.125 1.304
_________________________________________________________________

*Determination of enzymatic activities was effected according to
the standard method (T.K. Kirk, see above) that is based on the
oxidation of veratryl alcohol to aldehyde. Details in units/1
(oxidation of 1 ~mol veratryl alcohol to aldehyde/min.,
extinction increase at 308 nm).



The enzymes were extracted from the culture medium by
ultrafiltration (exclusion limit 10 kDa), dialysed against --
distilled water and then lyophilised.



Fractionation or cleaning of the crude enzyme preparations was
effected at preparative scale by liquid chromatography in an

anion exchanger matrix (Q-Sepharose, Pharmacia). Elutriation of
the individual fractions from the column was effected with a




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linear gradient of Na-acetate-buffer (10 mM to 600 mM) with a pH
value of 6.



Strains of Coriolus, Schizophyllum, Phlebia, Ganoderma, Fomes are
also suitable as additional white-rot fungi for obtaining
ligninases according to the present invention.




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