CA2006490A1 - Process for obtaining ligninolytic enzymes - Google Patents
Process for obtaining ligninolytic enzymesInfo
- Publication number
- CA2006490A1 CA2006490A1 CA 2006490 CA2006490A CA2006490A1 CA 2006490 A1 CA2006490 A1 CA 2006490A1 CA 2006490 CA2006490 CA 2006490 CA 2006490 A CA2006490 A CA 2006490A CA 2006490 A1 CA2006490 A1 CA 2006490A1
- Authority
- CA
- Canada
- Prior art keywords
- culture
- manganese dioxide
- added
- enzymes
- medium
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0065—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- General Chemical & Material Sciences (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
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.
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.
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 . ,,, , , . .: - , , ,~
, ' ' ,:, ; : ,, , .
Z00649~
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.
.. . .
; , , ,, ' . ::'' : , ' ~ ', :: ~ : . -: ' . .
' ' ' ,' ':,, , , ' ;', :~' ~, . , 200fi49C~
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):
.
', ~ : ' ' ' ~ . ' ' ' ' ' ' '' , , ' ', ' ' ' '" , .,' , , : :
": : ' .. .
-- X00~;49C~
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.
- :~ . ", : . . . . .
. .
', ,, : '', ,' , ' ''~ ,' .: ' ' ~ :' ,, ,, , ,,, , . :
" ,. . ..
.
.
.. . .. . ..
, -` 200~;~9n 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 . ~ ,, ,, , , , , , :
, X00~49C~
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.
.. . . . . . .
~ ~ . ~ , , ', . . .
" ' " ' ~, ' , ;' i ' ' '
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 . ,,, , , . .: - , , ,~
, ' ' ,:, ; : ,, , .
Z00649~
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.
.. . .
; , , ,, ' . ::'' : , ' ~ ', :: ~ : . -: ' . .
' ' ' ,' ':,, , , ' ;', :~' ~, . , 200fi49C~
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):
.
', ~ : ' ' ' ~ . ' ' ' ' ' ' '' , , ' ', ' ' ' '" , .,' , , : :
": : ' .. .
-- X00~;49C~
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.
- :~ . ", : . . . . .
. .
', ,, : '', ,' , ' ''~ ,' .: ' ' ~ :' ,, ,, , ,,, , . :
" ,. . ..
.
.
.. . .. . ..
, -` 200~;~9n 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 . ~ ,, ,, , , , , , :
, X00~49C~
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.
.. . . . . . .
~ ~ . ~ , , ', . . .
" ' " ' ~, ' , ;' i ' ' '
Claims (6)
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.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19883843381 DE3843381C1 (en) | 1988-12-23 | 1988-12-23 | |
| DEP3843381.8-41 | 1988-12-23 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2006490A1 true CA2006490A1 (en) | 1990-06-23 |
Family
ID=6369939
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2006490 Abandoned CA2006490A1 (en) | 1988-12-23 | 1989-12-22 | Process for obtaining ligninolytic enzymes |
Country Status (4)
| Country | Link |
|---|---|
| EP (1) | EP0374915A3 (en) |
| CA (1) | CA2006490A1 (en) |
| DE (1) | DE3843381C1 (en) |
| FI (1) | FI896259A0 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116219785A (en) * | 2023-02-27 | 2023-06-06 | 安徽金坤达包装集团有限公司 | Method for degrading lignin in situ by manganese oxide coupling laccase |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3843381C1 (en) * | 1988-12-23 | 1989-11-30 | Kernforschungsanlage Juelich Gmbh, 5170 Juelich, De | |
| CN100359324C (en) * | 2005-12-19 | 2008-01-02 | 北京化工大学 | Enzyme function sensitive membrane containing stripped MnO2 for biosensor and preparation method thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3843381C1 (en) * | 1988-12-23 | 1989-11-30 | Kernforschungsanlage Juelich Gmbh, 5170 Juelich, De |
-
1988
- 1988-12-23 DE DE19883843381 patent/DE3843381C1/de not_active Expired
-
1989
- 1989-12-21 EP EP19890123655 patent/EP0374915A3/en not_active Withdrawn
- 1989-12-22 CA CA 2006490 patent/CA2006490A1/en not_active Abandoned
- 1989-12-22 FI FI896259A patent/FI896259A0/en not_active Application Discontinuation
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116219785A (en) * | 2023-02-27 | 2023-06-06 | 安徽金坤达包装集团有限公司 | Method for degrading lignin in situ by manganese oxide coupling laccase |
Also Published As
| Publication number | Publication date |
|---|---|
| FI896259A0 (en) | 1989-12-22 |
| DE3843381C1 (en) | 1989-11-30 |
| EP0374915A3 (en) | 1991-05-08 |
| EP0374915A2 (en) | 1990-06-27 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FZDE | Dead |