CN1307127A - Method for producing lignoenzyme - Google Patents
Method for producing lignoenzyme Download PDFInfo
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- CN1307127A CN1307127A CN 00100711 CN00100711A CN1307127A CN 1307127 A CN1307127 A CN 1307127A CN 00100711 CN00100711 CN 00100711 CN 00100711 A CN00100711 A CN 00100711A CN 1307127 A CN1307127 A CN 1307127A
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Abstract
Lignoenzyme is produced by adding aqueous solution containing tween-80, sodium glutamate and magnesium sulfate to solid material comprising steam puffed cellulose material in 75-90 wt% and bran in 10-25 wt% with the solid to liquid ratio being 1 to 1.5-2.5 to obtain fermenting culture medium, pH regulation, disinfection, inoculation of liquid pure white rot funguses for fermentation; water leaching of the fermented solid matter and taking the suppernatant liquor as the lignoenzyme. The present invention has low production cost.
Description
The invention belongs to the using microbe field, particularly produce the method for lignoenzyme.
Xylogen is a renewable aromatics main on the earth.Estimate that 25% in the photosynthesis of plant institute fixed carbon is xylogen, so xylogen accounts for significant proportion in the energy that plant is caught from the sun.It is to be only second to cellulosic second affluent resources on the earth, yet, to it utilization research but seldom, be to wait the neck city [BusweLL, J.A.et aL., the comment of CRC biotechnology, 1987,6 (1): 1-10] developed in the natural polymer.Early eighties has been found in recent ten years, to have obtained certain progress in lignoenzyme and lignin biodegradation after lignin peroxidase (LiP) and the manganese peroxidase (MnP).But still there are many problems in the xylogen microbial transformation Study on degradation.(1) since the xylogen enzymolysis be a kind of non-narrow spectrum, be chain reaction process based on free radical, therefore, lignoenzyme is at chemical industry, coal chemistry and environment protection aspect have very big DEVELOPMENT PROSPECT; And the preparation method of existing lignoenzyme adopts synthetic medium mostly, make [Kirk, TK., et aL. with liquid fermentation method, enzyme and microbial technique, 1986,8:27-32], because of the essential Li Lu alcohol that adds costliness in this synthetic medium, and fermentation period is long, so production cost is very high, from practical application very big distance is arranged still, be badly in need of research high efficiency, low cost system.(2) research is to do the research of whiterot fungi degradation characteristic with the xylogen stand-in mostly, is difficult to reflect degradation process, and lignin biodegradation's mechanism is still unclear.
The objective of the invention is to overcome the high shortcoming of raw material costliness, production cost that prior art exists when producing lignoenzyme, a kind of method of new production lignoenzyme is provided, effectively utilize the cellulosic resource in the hope of reaching, and realize the purpose of its biomass complete utilization.
Technical scheme of the present invention is as follows: with the cellulose raw material of the quick-fried processing of vapour (with careless class for well) is main culture material, make the fermentation substratum by certain prescription, with the whiterot fungi is bacterial classification, mode with solid state fermentation is produced lignoenzyme, the lignoenzyme enzyme that is produced is lived higher, lignin peroxidase (Lip) is 2600U/L, it is 1100U/L that manganese relies on lignin peroxidase (Mnp), Lip and Mnp productivity have reached 21.667U/L.h and 11.458U/L.h (is raw material with the steam exploded wheat straw) respectively, overthrown common document [AddLeman, K., and ArchibaLd, F., applied environment microorganism, 1993,59:266-273] solid state fermentation of report produces the conclusion of lignoenzyme hardly.
The present invention produces the method for lignoenzyme, it is characterized in that this method is (weight percent): in the cellulose raw material and 10-25% wheat bran of the quick-fried processing of 75-90% vapour, add the aqueous solution and make fermention medium, solid-to-liquid ratio is (W/V) 1: 1.5-2.5, the wherein said aqueous solution contains the 0-0.2% tween 80, the 0-0.1% Sodium Glutamate, 0-0.1% sal epsom; With mineral acid adjust pH 4.5-6.5; Sterilization inserts the whiterot fungi liquid seeds that weighs 10% (V/W) with respect to the solid material, with ferment cellulose raw material after the quick-fried processing of vapour of solid state fermentation mode; After 5 hours, centrifugal removal solid substance, supernatant liquor are that gained enzyme liquid is lignoenzyme of the present invention with the lixiviate of solid state fermentation product adding distil water.
Said quick-fried broken processing is meant the cellulose raw material of the rough lumber of packing in steam-explosion jar, under the vapor pressure of 1.3-1.7MPa cellulose raw material was handled 5-15 minute, and instant decompression discharges; Said cellulose raw material is meant wheat straw, straw or cornstalk; Said whiterot fungi is meant Phanerochaete chrysosporium; Said solid state fermentation mode is meant tray formula solid state fermentation.
The selected steam puffed stalk of the present invention not only provides the nutritive substance such as the hemicellulose hydrolysate of easy utilization for whiterot fungi, and because xylogen partly is degraded, its degraded product has the material of inducing lignoenzyme to produce, and can replace the Li Lu alcohol of the essential costliness of adding of common liquid state fermentation fully.Owing to the fermentation raw material cheap and simple, needn't add expensive veratryl alcohol like this, and gained lignoenzyme product enzyme is alive high, the productive rate height, thereby, greatly reduce production cost.Make the suitability for industrialized production of lignoenzyme become a reality.
Below by embodiment and accompanying drawing technical scheme of the present invention is described further.
Fig. 1. process flow diagram of the present invention;
Fig. 2. the dynamic process synoptic diagram of the solid-state product enzyme of the present invention.
Number in the figure:
■.LiP????●.MnP
(1) in this technical process, steam explosion is handled and is meant that elder generation is long to 20-30mm with the raw material rough lumber, reinstalls steam-explosion jar, ties up down in 1.3-1.7MPa pressure and presses 5-15 minute, and instant decompression release promptly gets vapour and discloses.
(2) in this technical process, fermentation is meant the quick-fried fiber material of 75-90% vapour with culture medium prescription, and the 10-25% wheat bran adds the aqueous solution, and solid-to-liquid ratio is 1: 1.5-2.5.Wherein the aqueous solution contains the 0-0.2% tween 80, the 0-0.1% Sodium Glutamate, and 0-0.1% sal epsom is with mineral acid adjust pH 4.5-6.5.
(3) in this technical process, sterilising method is conventional steam sterilizing mode.
(4) in this technical process, bacterial classification is Phanerochaete chrysosporium (Phanerochate chrysosporiumEM-446).
(5) in this technical process, the method for preparing enzyme liquid is for the lixiviate of solid state fermentation product adding distil water after 5 hours, and centrifugal removal solid substance, supernatant liquor are gained enzyme liquid.
(6) in this technical process, enzyme activity determination is pressed document [Tiem, M., et aL., national science association journal, 1984,81:2280-2284.MichaeL, H.G., etaL., Enzymology method, 1988,161:258-264].In addition, disturb enzyme activity determination in order to eliminate pigment in the ultraviolet region, we have adopted reddish black orchid and phenol red in visible light mensuration again, come to the same thing.Embodiment 1: the steam exploded wheat straw solid state fermentation is produced lignoenzyme
1. steam exploded wheat straw
The wheat straw 80kg that rough lumber is grown to the about 25mm 1M that packs into
3In the steam-explosion jar, feed steam, treat in the drain tank to close the charging valve behind the freezing air, boost to 1.5MPa, dimension is pressed 10min, opens baiting valve, and instant decompression discharges and promptly gets steam exploded wheat straw.
2. the fermentation preparation and the sterilization (weight percent) of substratum
In quick-fried fiber material of 80% vapour and 20% wheat bran, add the aqueous solution, solid-to-liquid ratio 1: 2, wherein the aqueous solution contains 0.1% tween 80, and 0.05% L-glutamic acid and 0.05% sal epsom are with the hydrochloric acid adjust pH 4.5-6.5 of 0.1mol.In the large-scale sterilizing pot, sterilize, under 0.1MPa pressure, kept 50 minutes.
3. inoculation and sabot fermentation
Sterilized substratum is broken up, inserted Phanerochaete chrysosporium liquid seeds (heavily be with respect to the solid material 10% inoculum size) in sterilisable chamber, mixing, the sterilized enamel tray of packing into covers sterilized gauze above.Move into proving room, 39 ℃ leave standstill cultivation and got tunning in 5 days.
4. the preparation of enzyme liquid
Tunning adds 20 times distilled water lixiviate 5 hours, and the centrifugal solid substance of removing, supernatant liquor are lignoenzyme liquid.
5. utilize steam puffed stalk to produce the underlying condition conclusion of lignoenzyme method for fermenting raw materials.
(1). utilize steam puffed stalk to make fermentation raw material, the lignoenzyme that its solid state fermentation produces is lived and is higher than liquid state fermentation (table 1) far away.And in two kinds of fermentation modes, do not add Li Lu alcohol.The selected steam exploded wheat straw of this explanation not only provides the nutritive substance such as the hemicellulose hydrolysate of easy utilization for producing bacterium, and has the material of inducing lignoenzyme, can replace Li Lu alcohol fully.Like this, we just can produce lignoenzyme with simple culture material, and production cost is reduced greatly.
Table 1 utilizes the solid-state and liquid state fermentation lignoenzyme (39 ℃, 5 days) of steam puffed stalk
| ?LiP(U/L) | ?MnP(U/L) | |
| Liquid state fermentation | ??61.67 | ??27.12 |
| Solid state fermentation | ??365.12 | ??265.24 |
(2) owing to contain a certain amount of starch and VITAMIN in the wheat bran, help thalli growth, when adding wheat bran amount was big, mycelial growth was vigorous, and enzyme is alive minimum.The growth and the product enzyme relation of being inversely proportional to of whiterot fungi are described.Therefore, steam exploded wheat straw is 5: 1 with the ratio of wheat bran, and lignoenzyme is lived the highest.
The influence (39 ℃, 5d, liquor ratio 1: 2) that enzyme is produced in the different comparisons with wheat bran of table 2 steam exploded wheat straw
| Steam exploded wheat straw: wheat bran | ????2∶3 | ????3∶2 | ????4∶1 | ????5∶1 |
| ????LiP(u/L) | ???341.5 | ???380.76 | ???1156.3 | ??1836.78 |
| ????MnP(u/L) | ???615.31 | ???645.28 | ???878.24 | ??1263.42 |
(3) utilize the steam puffed stalk solid state fermentation to produce the lignoenzyme fermenting process as shown in Figure 2.The MnP enzyme produced early, had just reached peak (2600U/L) at the 4th day; And a product enzyme peak (1100U/L) appearred in LiP at the 5th day.LiP and MnP productivity have reached 21.667U/L.h and 11.458U/L.h respectively.
Embodiment 2: the quick-fried maize straw solid state fermentation of vapour is produced lignoenzyme
1. the quick-fried maize straw of vapour
The 80kg maize straw that rough lumber is grown to the about 25mm 1M that packs into
3In the steam-explosion jar, feed steam, treat in the drain tank to close the charging valve behind the freezing air, boost to 1.7MPa, dimension is pressed 5min, opens baiting valve, and instant decompression discharges and promptly gets steam exploded wheat straw.
2. the fermentation preparation and the sterilization (weight percent) of substratum
In quick-fried fiber material of 80% vapour and 20% wheat bran, add the aqueous solution, solid-to-liquid ratio 1: 2, wherein the aqueous solution contains 0.1% tween 80 and 0.05% L-glutamic acid, with the hydrochloric acid adjust pH 4.5-6.5 of 0.1mol.In the large-scale sterilizing pot, sterilize, under 0.1MPa pressure, kept 50 minutes.
3. inoculation and sabot fermentation
Sterilized substratum is broken up, inserted Phanerochaete chrysosporium liquid seeds (heavily be with respect to the solid material 10% inoculum size) in sterilisable chamber, mixing, the enamel tray of oneself sterilization of packing into covers sterilized gauze above.Move into proving room, 39 ℃ leave standstill cultivation 4-5 days.
4. the preparation of enzyme liquid
Tunning adds 20 times distilled water lixiviate 5 hours, and the centrifugal solid substance of removing, supernatant liquor are lignoenzyme liquid.Utilizing the quick-fried maize straw of vapour to produce lignoenzyme for raw material by solid fermentation lives: LiP and MnP are respectively 1534.08u/L and 2006.22u/L.
Embodiment 3: the steam exploded wheat straw solid state fermentation is produced lignoenzyme
1. steam exploded wheat straw
The 80kg wheat straw that rough lumber is grown to the about 25mm IM that packs into
3In the steam-explosion jar, feed steam, treat in the drain tank to close the charging valve behind the freezing air, boost to 1.3MPa, dimension is pressed 15min, opens baiting valve, and instant decompression discharges and promptly gets steam exploded wheat straw.
2. the fermentation preparation and the sterilization (weight percent) of substratum
In quick-fried fiber material of 90% vapour and 10% wheat bran, add the aqueous solution, solid-to-liquid ratio 1: 2, wherein the aqueous solution contains 0.1% tween 80, with the hydrochloric acid adjust pH 5.5 of 0.1mol.In the large-scale sterilizing pot, sterilize, under 0.01MPa pressure, kept 50 minutes.
3. inoculation and sabot fermentation
Sterilized substratum is broken up, inserted Phanerochaete chrysosporium liquid seeds (heavily be with respect to the solid material 10% inoculum size) in sterilisable chamber, mixing, the sterilized enamel tray of packing into covers sterilized gauze above.Move into proving room, 39 ℃ leave standstill cultivation 5 days.
4. the preparation of enzyme liquid
Tunning adds 20 times distilled water lixiviate 5 hours, and the centrifugal solid substance of removing, supernatant liquor are lignoenzyme liquid.Utilizing the quick-fried maize straw of vapour to produce lignoenzyme for raw material by solid fermentation lives: LiP and MnP are respectively 1485.00u/L and 1900.00u/L.Embodiment 4: the quick-fried maize straw solid state fermentation of vapour is produced lignoenzyme
1. the quick-fried maize straw of vapour
The 80kg maize straw that rough lumber is grown to the about 25mm 1M that packs into
3In the steam-explosion jar, feed steam, treat in the drain tank to close the charging valve behind the freezing air, boost to 1.5MPa, dimension is pressed 10min, opens baiting valve, and instant decompression discharges and promptly gets steam exploded wheat straw.
2. the fermentation preparation and the sterilization (weight percent) of substratum
In quick-fried fiber material of 80% vapour and 20% wheat bran, add the aqueous solution, solid-to-liquid ratio 1: 2.In the large-scale sterilizing pot, sterilize, under 0.01MPa pressure, kept 50 minutes.
3. inoculation and sabot fermentation
Sterilized substratum is broken up, inserted Phanerochaete chrysosporium liquid seeds (heavily be with respect to the solid material 10% inoculum size) in sterilisable chamber, mixing, the enamel tray of oneself sterilization of packing into covers sterilized gauze above.Move into proving room, 39 ℃ leave standstill cultivation 4-5 days.
4. the preparation of enzyme liquid
Tunning adds 20 times distilled water lixiviate 5 hours, and the centrifugal solid substance of removing, supernatant liquor are lignoenzyme liquid.Utilizing the quick-fried maize straw of vapour to produce lignoenzyme for raw material by solid fermentation lives: LiP and MnP are respectively 1600.00u/L and 2100.00u/L.
Claims (6)
1. method of producing lignoenzyme, it is characterized in that this method is (weight percent): in the cellulose raw material and 10-25% wheat bran of the quick-fried processing of 75-90% vapour, add the aqueous solution and make fermention medium, solid-to-liquid ratio is (W/V) 1: 1.5-2.5, the wherein said aqueous solution contains the 0-0.2% tween 80, the 0-0.1% Sodium Glutamate, 0-0.1% sal epsom; Adjust pH 4.5-6.5; Sterilization inserts the whiterot fungi liquid seeds, with ferment cellulose raw material after the quick-fried processing of vapour of solid state fermentation mode; The solid state fermentation product is added flooding and removes solid substance, and supernatant liquor is that gained enzyme liquid is lignoenzyme of the present invention.
2. the method for production lignoenzyme as claimed in claim 1, it is characterized in that said quick-fried broken processing is meant the cellulose raw material of the rough lumber of packing in steam-explosion jar, under the vapor pressure of 1.3-1.7MPa cellulose raw material was handled 5-15 minute, instant decompression discharges.
3. the method for production lignoenzyme as claimed in claim 1 or 2 is characterized in that said cellulose raw material is meant wheat straw, straw or cornstalk.
4. the method for production lignoenzyme as claimed in claim 1 is characterized in that said whiterot fungi is meant Phanerochaete chrysosporium.
5. the method for production lignoenzyme as claimed in claim 1 is characterized in that said solid state fermentation mode is meant tray formula solid state fermentation.
6. the method for production lignoenzyme as claimed in claim 1, the access amount that it is characterized in that said whiterot fungi liquid seeds for respect to the solid material heavy 10%.
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| CN 00100711 CN1307127A (en) | 2000-02-01 | 2000-02-01 | Method for producing lignoenzyme |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104389225A (en) * | 2014-11-06 | 2015-03-04 | 盐城师范学院 | Enhanced bioremediation method for pulping black liquor |
| CN105918614A (en) * | 2016-04-25 | 2016-09-07 | 陈华友 | Integration processing method of high-efficient corn straw biological feed |
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2000
- 2000-02-01 CN CN 00100711 patent/CN1307127A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104389225A (en) * | 2014-11-06 | 2015-03-04 | 盐城师范学院 | Enhanced bioremediation method for pulping black liquor |
| CN105918614A (en) * | 2016-04-25 | 2016-09-07 | 陈华友 | Integration processing method of high-efficient corn straw biological feed |
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