Microbial method for producing lignin peroxidase

Method for producing lignin-peroxydase from the fungus Phanerochaete chrysosporium. The method comprises a first step of culture wherein the phospholipids and the emulsified fatty acids are added to the culture medium; a second step during which veratrylic alcohol is added to the culture medium, the culture medium being partially renewed for the second culture step and totally renewed for the third and fourth culture steps, by varying the content of constituents in said medium. Application to the production of lignin-peroxydase with important yields.

The present invention relates to an improved method of producing 
ligninolytic enzyme or lignin peroxidase. 
BACKGROUND OF THE INVENTION 
In SCIENCE, (1983) 221, pp. 661-663, MING TIEN and T. KENT KIRK have 
described an extracellular enzyme produced by Phanerochaete chrysosporium 
Burdsall, which is a basidiomycetes fungus; in the presence of hydrogen 
peroxide, this enzyme is capable of causing the oxidizing degradation of 
various model compounds having lignin substructures, as well as the 
degradation of fir or birch lignin. 
This enzyme, called "lignin peroxidase", has been characterized [M. TIEN 
and T. K. KIRK, PROC. NATL. ACAD. SCI. USA, (1984) vol. 81, pp. 2280-2284] 
as being a glycoprotein of about 42 kDa. 
French patent 2 574 427 claims two novel strains of Phanerochaete 
chrysosporium Burdsall which are capable of developing a particularly high 
ligninolytic activity in media of unlimited nitrogen content, whereas the 
previously known strains produced the ligninolytic enzyme only in a 
nitrogen-deficient culture medium, the biodegradation process which 
produced this enzyme also being very slow under these conditions. 
According to said patent, the culture medium suitable for promoting the 
production of lignin peroxidase contains a source of assimilable nitrogen 
which can be asparagine, ammonium nitrate or ammonium tartrate; it also 
contains a source of assimilable carbon such as glucose, mannose, starch, 
melibiose, mannitol, xylose, maltose, adonitol, arabitol, fructose, 
sorbitol, raffinose, xylitol, D(+)-trehalose or glycerol, the last of 
these being preferred; it further contains a source of assimilable mineral 
salts such as iron citrate, KH.sub.2 PO.sub.4, ZnSO.sub.4, MnSO.sub.4, 
CaCl.sub.2, CuSO.sub.4, NaCl, FeSO.sub.4, CoSO.sub.4, AlK(SO.sub.4).sub.2, 
H.sub.3 BO.sub.3, Na.sub.2 MoO.sub.4 or MgSO.sub.4. 
French patent 2 600 077 proposes significantly increasing the production 
yields of lignin peroxidase by cultivating the fungus Phanerochaete 
chrysosporium on a base culture medium supplemented with a stimulant 
selected from unsaturated fatty acids, natural amino acids and mixtures 
thereof. The preferred unsaturated fatty acids are oleic acid, linoleic 
acid, palmitoleic acid and arachidonic acid, preferably in emulsified 
form, and the preferred natural amino acids are serine, threonine, 
isoleucine, glycine, valine and tyrosine, but more particularly 
isoleucine, serine, threonine and glycine. The optimum concentration of 
oleic acid is about 800 mg/liter or, in the case of emulsified oleic acid, 
about 400 mg/liter; the optimum concentration of isoleucine is about 6.5 
mg/liter. 
It should be pointed out that JAGER et al. (APPLIED AND ENVIRONMENTAL 
MICROBIOLOGY, Nov. 1985, pp. 1274-1278) have proposed adding a detergent, 
such as TWEEN 80 or TWEEN 20, to the culture medium of the fungus, in 
submerged cultures, in order to increase the production of lignin 
peroxidase in proportions comparable to that currently obtained in 
stationary cultures. FAISON & KIRK [APPL. ENVIRON. MICROBIOL., (1985) 49, 
pp. 299-304] and LEISOLA et al. [J. BIOTECHNOL., (1985) 3, pp. 97-107] 
have reported that the addition of veratryl alcohol, which is a secondary 
metabolite of Phanerochaete chrysosporium, increases the synthesis of 
lignin peroxidase. 
In an article published in ENZ. MICROBIAL TECHNOL., (1987) 9, pp. 245-249, 
ASTHER et al. have shown that, in the presence of exogenous oleic acid 
emulsified by TWEEN 80, there is a significant increase in the production 
of lignin peroxidase and a considerable reduction in the fermentation time 
required to reach the maximum enzymic activity. 
In a more recent article, published in APPL. MICROBIOL. BIOTECHNOL., (1988) 
27, pp. 393-398, ASTHER et al. have reported that the production of lignin 
peroxidase by the fungus Phanerochaete chrysosporium is further increased 
when olive oil supplemented with soya azolectin, which constitutes a 
source of phospholipids, is added to the culture medium. 
In a communication delivered at the Annual Meeting of the ASM which took 
place from May 8 to 13, 1988 in Miami, Fla., United States, ASTHER et al. 
reported that a maximum production of lignin peroxidase is obtained from 
cultures of Phanerochaete chrysosporium when culture is carried out at two 
different temperatures, namely at 37.degree. C. during the growth phase of 
the mycelium, for the first two days of incubation, and then at 30.degree. 
C. during the lignin peroxidase production phase. 
LEISOLA et al. [q.v. the above-cited article published in J. BIOTECHNOL. in 
1985] have identified four hemes containing lignin peroxidase activity in 
extracellular fluid of three-day-old cultures of Phanerochaete 
chrysosporium BKM-F-1767, induced by veratryl alcohol. JAGER et al. [APPL. 
ENVIRON. MICROBIOL., (1985) 50, pp. 1274-1278] have separated 8 peaks with 
an absorbance at 409 nm (hemoproteins), assuming that relatively minor 
differences between the profiles of the proteins simply reflect the age of 
the cultures. 
OBJECTS OF THE INVENTION 
The aim of the present invention was to provide a method of producing 
lignin peroxidase from Phanerochaete chrysosporium under controlled 
temperature conditions which are different during the growth phase of the 
mycelium and during the actual lignin peroxidase production phase, and 
which make it possible to favour the production of the hemoproteins having 
the maximum lignin peroxidase activity, at the expense of biomass 
production. 
SUMMARY OF THE INVENTION 
The present invention relates to a method of producing lignin peroxidase 
from a fungus known as Phanerochaete chrysosporium, in a culture medium 
containing activators of the enzyme, such as phospholipids and veratryl 
alcohol, which method is characterized in that it comprises: 
a first step involving the culture of cells of Phanerochaete chrysosporium 
for an incubation period of about 2 days, in a synthetic culture medium 
comprising salts of potassium, calcium and magnesium, trace elements 
(iron, zinc, manganese, copper), an appropriate source of nitrogen, a 
source of carbon advantageously consisting of glycerol, yeast extract, a 
source of phospholipids and a source of emulsified fatty acids; 
a second step involving culture of the mycelium formed during the first 
step, for a period of about 3 days (from D3 to D5), in a culture medium 
which has been partially renewed (advantageously to the extent of about 
15%), to which veratryl alcohol--an activator and protector of lignin 
peroxidase production--has been added, and whose content of phospholipids 
has been reduced to about 1/7-1/8 of what it was in the culture medium of 
the first step, the present culture medium not containing emulsified fatty 
acids; 
a third step involving culture at the optimum lignin peroxidase production, 
i.e. after about 5 days, during which the whole of the culture medium of 
the second step is replaced with a synthetic culture medium analogous to 
that of the first step, which contains the same proportion of 
phospholipids and veratryl alcohol as the culture medium of the second 
step, and in which the yeast extract, the source of nitrogen and the 
source of carbon--advantageously consisting of glycerol--have been reduced 
to 1/4 of their content in the culture medium of the first step, these 
three components together forming a partial regeneration medium for the 
biomass; 
a fourth step consisting in continuing the culture with non-proliferating 
cells for about 8 days (from D6 to D13), the culture medium being totally 
renewed every 24 hours and replaced with a medium not containing yeast 
extract, emulsified fatty acids or glycerol, but containing the activators 
and protectors of the enzyme production, consisting of veratryl alcohol 
and phospholipids, in the same proportions as the culture media of the 
second and third steps; and 
a fifth step involving separation of the enzyme produced and, if necessary, 
purification of said enzyme. 
The precise strains of the white rot fungus Phanerochaete chrysosporium 
Burdsall employed in this invention have been deposited under the Budapest 
Treaty at the Collection Nationale de Cultures de Microorganisms (CNCM) at 
the Pasteur Institute, 25 Rue de Dr. Roux, Paris 75015 France, and are 
available to the public. These strains of Phanerochaete chrysosporium 
Burdsall bear the identifying numbers CNCM I-398 and CNCM I-399. These 
same strains and methods for producing them are also described and claimed 
in European Patent No. 188,931. Another designation at the CNCM, Institute 
Pasteur, Paris for CNCM I-398 is INA-12, as disclosed by Applicants in 
Enzyme Microbial Technology, Vol. 13, September 1991 at pp. 727-733. 
In one advantageous mode of carrying out the method of producing lignin 
peroxidase according to the present invention, the first step involving 
culture of the cells takes place at an incubation temperature of about 
37.degree. C., whereas the next steps are carried out at incubation 
temperatures of about 30.degree. C. 
In another advantageous mode of carrying out the method according to the 
present invention, the first step involving culture of the cells takes 
place in a culture medium buffered at pH 6.5, whereas the next steps are 
carried out in a culture medium buffered at pH 5.5. 
In yet another advantageous mode of carrying out the method of producing 
lignin peroxidase according to the present invention, the source of 
phospholipids advantageously consists of soya azolectin. 
In another advantageous mode of carrying out the method of producing lignin 
peroxidase according to the present invention, the culture medium of the 
first step has a content of nitrogen source of the order of 1.84 g/liter 
for a content of carbon source (glycerol) of the order of 10 g/liter, for 
a content of phospholipids (soya azolectin) of the order of 0.75 g/liter 
and for a content of yeast extract of 1 g/liter. 
In yet another advantageous mode of carrying out the method of producing 
lignin peroxidase according to the present invention, the culture medium 
of the second step differs from the culture medium of the first step by 
its pH (5.5), its reduced content of phospholipids (of the order of 0.1 
g/liter) and its content of veratryl alcohol (of the order of 2.5 mM). 
In another advantageous mode of carrying out the method of producing lignin 
peroxidase according to the present invention, the culture medium of the 
third step differs from the culture medium of the second step by its 
reduced content of glycerol (about 2.5 g/liter), nitrogen source (in 
particular about 0.46 g/liter of diammonium tartrate) and yeast extract 
(0.25 g/liter). 
In another advantageous mode of carrying out the method of producing lignin 
peroxidase according to the present invention, the culture medium of the 
fourth step differs from the culture medium of the third step in that it 
does not contain yeast extract, a source of nitrogen or a source of 
carbon. 
In another advantageous mode of carrying out the method of producing lignin 
peroxidase according to the invention, the third and fourth steps are 
carried out continuously. 
The method according to the present invention affords a considerable 
increase in the lignin peroxidase productivity and the lignin peroxidase 
activity compared with all the methods known hitherto. In fact, whereas a 
lignin peroxidase activity of the order of 22.4 nKat.ml.sup.-1 is obtained 
in the case where a culture medium of Phanerochaete chrysosporium is 
supplemented with oleic acid emulsified with Tween 80 at a concentration 
of 0.04% (weight/volume), and whereas the lignin peroxidase activity is of 
the order of 33.3 nKat.ml.sup.1 when the culture medium is supplemented 
with azolectin as the source of phospholipids and olive oil as the source 
of lipids, the lignin peroxidase activity can reach 40.6 nKat.ml.sup.-1 
when using the method according to the present invention, in which culture 
media supplemented successively with phospholipids and emulsified fatty 
acids, and then with veratryl alcohol and phospholipids, are used at 
different incubation temperatures of 37.degree. C. for the first culture 
medium and 30.degree. C. for the second. Moreover, whereas the lignin 
peroxidase productivity is of the order of 40.6 nKat.ml.sup.-1.day.sup.-1 
when using the method according to the invention, it is as low as 4.5 
nKat.ml.sup.-1.day.sup.-1 when the culture medium contains oleic acid 
emulsified with Tween 80, it is 8.3 nKat.ml.sup.-1.d.sup.-1 when the 
culture medium contains azolectin and olive oil and it is 1.6 
nKat.ml.sup.-1.day.sup.-1 when the culture medium contains veratryl 
alcohol 1 mM, with an incubation temperature of 39.degree. C., as 
described in the prior art cited. 
The FPLC (fast protein liquid chromatography) profiles of extracellular 
proteins obtained from cultures incubated at 37.degree. C. for the first 
two days and then at 30.degree. C., with the changes of culture media 
according to the present invention, show the presence of several 
hemoproteins, nine of which have lignin peroxidase activity, with a large 
peak n.degree. 4. 
In addition to the foregoing provisions, the invention also includes other 
provisions which will become apparent from the following description. 
PREFERRED EMBODIMENTS OF THE INVENTION 
The invention will be understood more clearly with the help of the 
following additional description referring to the attached drawings, in 
which:

EXAMPLE 1 OF LIGNIN PEROXIDASE PRODUCTION 
The microorganism used is the strain Phanerochaete chrysosporium INA-12 
(CNCM n.degree. I-398). 
Cells of Phanerochaete chrysosporium INA-12 immobilized on polyurethane 
foam are treated in the following manner: 
1. 6.times.10.sup.7 conidiospores are inoculated into a culture medium of 
the following composition: 
______________________________________ 
Solution of salts 
Potassium dihydrogen phosphate KH.sub.2 PO.sub.4 
2 g/l 
Calcium chloride CaCl.sub.2.2H.sub.2 O 
0.14 g/l 
Magnesium sulphate MgSO.sub.4.7H.sub.2 O 
0.7 g/l 
Solution of trace elements 
Iron (ferrous) sulphate FeSO.sub.4.7H.sub.2 O 
0.07 g/l 
Zinc sulphate ZnSO.sub.4.7H.sub.2 O 
0.0462 g/l 
Manganese sulphate MnSO.sub.4.H.sub.2 O 
0.035 g/l 
Copper sulphate CuSO.sub.4.5H.sub.2 O 
0.007 g/l 
Solution of vitamins 
Thiamine hydrochloride (vitamin B.sub.1) 
0.0025 g/l 
Source of nitrogen 
Ammonium tartrate 1.84 g/l 
Source of carbon 
Glycerol 10 g/l 
Yeast extract 1 g/l 
Soya azolectin (phospholipids) 
0.75 g/l 
Oleic acid emulsified with Tween 80 
0.4 g/l 
pH of the culture: 6.5 (the buffer used is 
2,2-dimethylsuccinic acid) 
______________________________________ 
INTERMEDIATE TEMPERATE: 37.degree. C. 
After 100% O.sub.2 has been blown in for two minutes, culture is carried 
out, without agitation, in 150 ml Erlenmeyer flasks containing 30 ml of 
the abovementioned aqueous culture medium, from D0 to D2 at 37.degree. C. 
2. 15% of the above-mentioned total medium is withdrawn and replaced with a 
culture medium such as described in 1. above, except as regards the yeast 
extract, the soya azolectin and the emulsified oleic acid, the first and 
third of which are omitted, as are the glycerol, the diammonium tartrate, 
the salts, the trace elements and the vitamins, while the content of soya 
azolectin in the medium is reduced by a factor of 7.5 (0.1 g/liter) and 
veratryl alcohol (2.5 mM) is added to said medium, the veratryl alcohol 
and the soya azolectin acting as inductors of lignin peroxidase 
production. Culture is continued from D3 to D5 inclusive at an incubation 
temperature of 30.degree. C., which is the optimum temperature for lignin 
peroxidase production. 
3. On D5, the whole of the culture medium is withdrawn and replaced with a 
culture medium of the following composition: 
______________________________________ 
Source of nitrogen 
Diammonium tartrate 0.46 g/l 
Source of carbon 
Glycerol 2.5 g/l 
Yeast extract 0.25 g/l 
Soya azolectin 0.1 g/l 
Veratryl alcohol 2.5 mM 
______________________________________ 
The veratryl alcohol and the soya azolectin present in this medium act as 
inductors and protectors of the lignin peroxidase, as in the culture 
medium used in step 2. above, and the glycerol, the ammonium tartrate and 
the yeast extract together form a regeneration medium for the biomass. The 
pH of the culture medium is adjusted to 5.5 and the incubation temperature 
is 30.degree. C. The glycerol, the source of nitrogen and the yeast 
extract act on the production of biomass and not on the production of 
enzyme, which is why they are present in the regeneration medium. However, 
their concentrations are reduced to 25% of what they are in the culture 
medium of step 1. above, so as to limit the production of biomass in 
proportions which do not jeopardize production of the enzyme. 
4. From D6, the whole of the culture medium is withdrawn every 24 hours and 
replaced with a culture medium of the same composition as that used in 
step 2. above, and culture is continued up to D13 at 30.degree. C. and at 
pH 5.5. 
FIG. 1 attached represents a diagram of the method of producing lignin 
peroxidase by using non-proliferating cells of Phanerochaete 
chrysosporium, according to the present invention. 
In this diagram: 
______________________________________ 
##STR1## 
represents the temperature profile, 
##STR2## 
represents the replacement of 15% of the culture medium with a 
culture medium comtaining 0.1 g/l of azolectin and 2.5 mM veratryl 
alcohol, 
##STR3## 
represents the replacement of 100% of the culture medium with a 
culture medium containing azolectin (0.1 g/l), veratryl alcohol 
(2.5 mM) and a partial regeneration medium for the biomass 
(glycerol 2.5 g/l, ammonium tartrate 0.46 g/l, yeast extract 0.25 
g/l), and 
##STR4## 
represents the replacement (every 24 hours) of 100% of the 
culture medium with a culture medium containing azolectin (0.1 
g/l) and veratryl alcohol (2.5 mM). 
______________________________________ 
5. The cultures obtained are harvested and filtered on a glass fibre 
filter. The supernatant is dialyzed overnight against distilled water. The 
extracellular medium is then concentrated to 1/10 of its initial volume by 
ultrafiltration through an Amicon YM10 membrane. 
The proteins contained in the concentrated fluid are determined by FPLC 
(fast protein liquid chromatography) using a PHARMACIA LCC 500 
chromatograph equipped with a Mono QHR 515 anion exchange column, with a 
gradient of NaCl in 10 mM sodium cacodylate (pH 5.9). The FPLC profiles of 
the isoenzymes of lignin peroxidase obtained are shown in FIG. 2 attached, 
in which 
FIG. 2A represents the profiles of extracellular proteins of Phanerochaete 
chrysosporium INA-12 under standard culture conditions without renewal of 
the culture medium, the incubation temperature being 37.degree. C.; 
FIG. 2B represents said protein profiles obtained with renewal of the 
atmosphere (100% oxygen) after two days of culture; 
FIG. 2C represents said protein profiles obtained with renewal of the 
atmosphere with oxygen and changing of 15% of the medium with 2.5 mM 
veratryl alcohol after two days of culture; 
FIG. 2D represents said protein profiles obtained with renewal of the 
atmosphere with oxygen and changing of 15% of the medium with 2.5 mM 
veratryl alcohol and 0.1 g/l of azolectin after two days of culture; and 
FIG. 2E represents said protein profiles obtained with renewal of the 
atmosphere with oxygen and changing of 15% of the medium with 2.5 mM 
veratryl alcohol and 0.1 g/l of azolectin, and changing of the incubation 
temperature from 37.degree. C. to 30.degree. C. after two days of culture. 
The absorbance at 405 nm is represented by solid lines and the absorbance 
at 280 nm by broken lines; the sloping line represents the gradient of 
NaCl. 
These profiles show that the extracellular fluid contains several 
hemoproteins, six of which have lignin peroxidase activity; these 
hemoproteins correspond to peaks 1, 2, 3, 4, 5, 6, 7, 8 and 9. 80% of the 
lignin peroxidase activity is associated with peaks 7, 8 and 9. The 
cultures whose incubation temperatures were successively 37.degree. C. and 
then 30.degree. C., and those whose incubation temperature was 30.degree. 
C. only, show an increase in the proportion of peak 4. 
EXAMPLE 2 OF LIGNIN PEROXIDASE PRODUCTION 
The conditions of Example 1 are repeated, 100 ml of medium being introduced 
into a 250 ml Erlenmeyer flask; an enzyme production of 46.4 nKat/ml is 
obtained. 
EXAMPLE 3 OF LIGNIN PEROXIDASE PRODUCTION 
The microorganism used is also the strain Phanerochaete chrysosporium 
INA-12 (CNCM n.degree. I-398). 
Cells of Phanerochaete chrysosporium INA-12 immobilized on polyurethane 
foam are treated as in step 1 of Example 1. 
The reactor is inoculated with 2.10.sup.5 spores/ml. There is direct 
immobilization in situ in the reactor. 
The medium is fed continuously with oxygen introduced through a central 
shaft, where the agitating turbine (200 rpm) is also situated. The O.sub.2 
concentration is regulated to about 60% of saturation of the medium with 
air. The cells of Phanerochaete chrysosporium immobilized on the 
polyurethane foam are placed at the periphery of the bioreactor, around 
the central shaft. 
Steps 2 to 5 are identical to those of Example 1. 
The enzyme titre obtained reaches 25 nKat/ml/day. 
The method according to the present invention makes it possible to control 
the production of lignin peroxidase and consequently affords the 
possibility of producing this enzyme on an industrial scale. 
As is apparent from the foregoing description, the invention is in no way 
limited to those modes of execution, embodiments and modes of application 
which have now been described more explicitly; on the contrary, it 
encompasses all the variants thereof which may occur to those skilled in 
the art, without deviating from the framework or the scope of the present 
invention.