CN105886480B - A kind of whiterot fungi secretion manganese peroxidase culture medium and its application - Google Patents
A kind of whiterot fungi secretion manganese peroxidase culture medium and its application Download PDFInfo
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Abstract
The invention discloses a kind of whiterot fungi secretion manganese peroxidase culture mediums and preparation method thereof, the culture medium is low nitrogen asparagine-succinic acid culture medium, the culture medium includes that carbon source concentration is 5~30g/L, nitrogen concentration is 5~30mmol/L, manganese ion concentration is 26.7~200 μm of ol/L, Tween 80 concentration is 0.25~0.75mL/L, MgSO4﹒ 7H2O concentration is 0.1~0.4g/L, and mineral element solution additional amount is 10~20mL/L, medium pH value 3.5~5.5.The present invention carries out biodegrade to lignin using the lignin-degrading enzymes manganese peroxidase in whiterot fungi, finally lignin can be thoroughly degraded to CO2And H2O just fundamentally solves the problem of environmental pollution of paper industry.Manganese peroxidase can be with lignin degrading and phenol type substances, and can also attack various organic pollutants includes persistent chemical pesticide, dye decolored etc..
Description
Technical field
The present invention relates to a kind of manganese peroxidase culture mediums more particularly to a kind of whiterot fungi to secrete manganese peroxidase
Culture medium and its application.
Background technique
Lignin is the natural polymer organic matter in plant kingdom next in number only to cellulose, be it is the most abundant, can be from
The aromatic compound obtained in renewable resource.The biodegrade of lignin plays a key role in earth carbon cycle.
There is the microorganisms of many lignin degradings for nature.They can attack lignin degrading and cause timber white rot sick,
It can generate some extracellular oxidative systems in lignin lumen, mainly include manganese peroxidase (Manganese
Peroxidases/MnPs), laccase (Laccases/Lac) and lignin peroxidase (Lignin peroxidases/
LiPs), they are the chief components of whiterot fungi Ligninolytic Enzymes, and wherein MnP is a kind of dependence H2O2Ferrous iron it is blood red
Plain glucoproteinase is a kind of peroxidase of most common lignin degrading, in the non-specific born of the same parents of Fungi Degrading Lignin
In external oxidation reductase, MnP is played a crucial role.
Though manganese peroxidase has so important purposes, its application in production practice is still extremely limited, main
It wants to cause manganese peroxidase production of enzyme not high the reason is that the activity of most production bacterial strain synthesis manganese peroxidase is low.Therefore, high
The acquisition for producing manganese peroxidase bacterial strain is environmentally protective to regenerated resources, the saving energy, or from comprehensive treatment water pollution, exploitation
New material, which is all one, far-reaching social effect and urgent realistic meaning and the research class with potential economic value
Topic.
Summary of the invention
It is an object of the invention to overcome the deficiencies of the prior art and provide a kind of secretion manganese peroxidase trainings of whiterot fungi
Base and preparation method thereof is supported, it can be achieved that the industrialized production of manganese peroxidase.
The present invention is achieved by the following technical solutions, and culture medium of the present invention is low nitrogen asparagine-succinic acid
Culture medium, the culture medium include that carbon source concentration is 5~30g/L, and nitrogen concentration is 5~30mmol/L, and manganese ion concentration is
26.7~200 μm of ol/L, Tween 80 concentration are 0.25~0.75mL/L, MgSO47H2O concentration is 0.1~0.4g/L, mineral
Element Solution additional amount is 10~20mL/L, medium pH value 3.5~5.5.
As one of preferred embodiment of the invention, the carbon source appointing in bagasse, glucose, straw, cornstarch
It is a kind of.
As one of preferred embodiment of the invention, the nitrogen source is in ammonium tartrate, peptone, ammonium sulfate, dregs of beans
It is any.
The culture medium includes that concentration of glucose is 5g/L, and peptone concentration 20mmol/L, manganese ion concentration is 200 μ
Mol/L, Tween 80 concentration are 0.25mL/L, MgSO47H2O concentration is 0.3g/L, and mineral element solution additional amount is 15mL/
L, medium pH value 4.5.
The mineral element solution of every 100mL includes ironic citrate hydrate 0.0087g, ZnSO4·7H2O 0.01g,
MnSO4·H2O 0.045g, CoCl2·6H2O 0.01g, CuSO4·5H2O 0.001g。
It further include a variety of vitamin solution in the culture medium, more kinds of vitamin solution of every 100mL include D biotin
0.002g, folic acid 0.0002g, vitamin B1 0.005g, vitamin B2 0.0005g, vitamin B6 0.001g, vitamin B12
0.00001g, niacin 0.0005g, pantothenate 0.0005g, p-aminobenzoic acid 0.0005g, DL-6,8- lipoic acid 0.0005g.
The whiterot fungi is Hericium erinaceus Hericium erinaceum.
A kind of preparation method of whiterot fungi secretion manganese peroxidase culture medium, comprising the following steps:
(1) preparation solid medium PDA, including 180~220g of potato, 18~22g of glucose, 18~22g of agar powder,
3~8g of peptone, distilled water are settled to 1000mL, and pH value is natural；
(2) side length 8~10mm fungus block is made in activated whiterot fungi strain on PDA plate, obtained fungus block is connect
Enter in low nitrogen asparagine-succinic acid fluid nutrient medium；
It (3) is 100mL bottling, 8 fungus blocks of every bottle of inoculation, then in 100~300r/min of revolving speed, temperature according to liquid amount
Constant-temperature shaking culture 7d in 20~36 DEG C of shaking tables, is made mother liquor.
As one of preferred embodiment of the invention, (1) prepares solid medium PDA, including potato 200g, glucose
20g, agar powder 20g, peptone 5g, distilled water are settled to 1000mL, and pH value is natural；
(2) 8~10mm fungus block is made in activated whiterot fungi strain on PDA plate, obtained fungus block is accessed low
In nitrogen asparagine-succinic acid fluid nutrient medium；
It (3) is that 100mL bottles according to liquid amount, 8 fungus blocks of every bottle of inoculation, then in revolving speed 200r/min, 28 DEG C of temperature
Constant-temperature shaking culture 7d in shaking table, is made mother liquor.
The present invention has the advantage that the present invention using the lignin-degrading enzymes manganese peroxide in whiterot fungi compared with prior art
Compound enzyme carries out biodegrade to lignin, finally lignin can be thoroughly degraded to CO2And H2O is just fundamentally solved and is made
The problem of environmental pollution of paper industry.Manganese peroxidase can also be attacked various organic with lignin degrading and phenol type substances
Pollutant includes persistent chemical pesticide, dye decolored etc., the method for fermenting and producing manganese peroxidase of the invention, most
High manganese peroxidase enzyme activity is up to 400.6U/L, about 10.4 times before optimization；It is tested using 5L fermentor, highest manganese peroxide
Compound enzyme activity is 1.37 times under the conditions of shaking flask up to 531.8U/L, about yield of enzyme.
Detailed description of the invention
Fig. 1 is the tendency chart that Hericium erinaceus produces manganese peroxidase first time orthogonal test carbon source kind；
Fig. 2 is the tendency chart that Hericium erinaceus produces manganese peroxidase first time orthogonal test carbon source concentration；
Fig. 3 is the tendency chart that Hericium erinaceus produces manganese peroxidase first time orthogonal test nitrogen source type；
Fig. 4 is the tendency chart that Hericium erinaceus produces manganese peroxidase first time orthogonal test nitrogen concentration；
Fig. 5 is the tendency chart that Hericium erinaceus produces manganese peroxidase first time orthogonal test manganese ion concentration；
Fig. 6 is that Hericium erinaceus produces second of orthogonal test liquid amount tendency chart of manganese peroxidase；
Fig. 7 is that Hericium erinaceus produces second of orthogonal test Tween 80 tendency chart of manganese peroxidase；
Fig. 8 is that Hericium erinaceus produces second of orthogonal test pH value tendency chart of manganese peroxidase；
Fig. 9 is that Hericium erinaceus produces second of orthogonal test MgSO of manganese peroxidase4﹒ 7H2O tendency chart；
Figure 10 is that optimization culture Hericium erinaceus produces manganese peroxidase curve graph.
Specific embodiment
It elaborates below to the embodiment of the present invention, the present embodiment carries out under the premise of the technical scheme of the present invention
Implement, the detailed implementation method and specific operation process are given, but protection scope of the present invention is not limited to following implementation
Example.
1, the measuring method of manganese peroxidase enzyme activity
Manganese peroxidase vigour-testing method uses 2,6- syringol method.Contain 50 μ L in 1mL reaction system
The reaction substrate 2 of (10mmol/L), 6- syringol (2,6-DMP), 840 μ L of sodium malonate buffer (50mmol/L),
MnSO4(10mmol/L) 50 μ L, H2O2The enzyme solution that (10m mol/L) 10 μ L starting reaction and 50 μ L have diluted, 25 DEG C of starting reactions
And the value added △ OD470 for reacting reaction solution absorbance at 470nm in preceding 1min is measured, using the enzyme solution of inactivation as blank.
Definition is under this condition, it is 1 enzyme activity unit (U/ that every min, which is catalyzed enzyme amount needed for 1 μm of 2,6-syringol of ol aoxidizes,
L)。
And manganese peroxidase activity value is calculated as follows:
Manganese peroxidase enzyme activity (UL–1)=[106× V1 × △ A × N]/[V2 × ε × △ t],
In formula: V1-reaction total volume；V2-enzyme solution volume；N-enzyme solution extension rate；ε-molar extinction coefficient；ε
470=49600molL–1·cm–1；△ t-reaction time；The incremental value of △ A reaction time internal absorbance.
Manganese peroxidase oxydasis Mn2+For Mn3+Mn3+Molar absorptivity of the compound formed with sodium malonate at 470nm
Coefficient is 49600mol/L/cm.
2, the optimization of culture medium
The culture medium of whiterot fungi secretion manganese peroxidase optimizes, and the factor level of the 1st orthogonal test is 4
A grade.4 kinds of carbon sources, 4 kinds of nitrogen sources and manganese ion have been selected, has included: A carbon source kind, B carbon source concentration using 5 kinds of experimental factors
(g/L), C nitrogen source type, D nitrogen concentration (mmol/L) and E Mn2+Concentration (μm olL).Whiterot fungi secretes manganese peroxidase
The culture medium of enzyme optimizes, and the factor level of the 1st orthogonal test is 4 grades.It include: A using 5 kinds of experimental factors
4 kinds of carbon source kinds include bagasse, glucose, straw, cornstarch；B carbon source concentration is respectively 5~30g/L；4 kinds of nitrogen source kinds of C
Class includes ammonium tartrate, peptone, ammonium sulfate, dregs of beans；D nitrogen concentration is respectively 5~30mmol/L；E manganese ion is concentration
26.7~200 μm of ol/L.The 1st orthogonal test L is designed according to biostatistics16(45) orthogonal arrage, producing enzyme basis train
On the basis of supporting base, the first time orthogonal test of composition 16 groups of different formulations culture mediums of progress of changing section culture medium, every group
3 repetitions are done in test, need to be extracted the sample of 48 parts of enzymes every time and be measured enzyme activity.
Influence of the first time orthogonal test to MnP enzyme is produced
The factor level of first time orthogonal test be 4 grades, select 4 kinds of carbon sources and 4 kinds of nitrogen sources, using 5 kinds test because
Element includes: A carbon source kind, B carbon source concentration (g/L), C nitrogen source type, D nitrogen concentration (mmol/L) and E Mn2+Concentration (μ
Mol/L), it is shown in Table 1.
1 Hericium erinaceus of table produces the design of MnP first time orthogonal test factor level
The 1st orthogonal test L is designed according to biostatistics16(45) orthogonal arrage, in the base of producing enzyme basal medium
On plinth, the composition of changing section culture medium carries out the first time orthogonal test of 16 groups of different formulations culture mediums, and every group of test does 3
It repeats, the sample of 48 parts of enzymes need to be extracted every time and measure enzyme activity, mainly investigate the situation that every kind of factor produces MnP, the results are shown in Table 2.
Compare each factor level Largest Mean ki, finally show that the optimization formula of culture medium is A2B1C2D3E4, i.e., carbon source is glucose
Concentration is 5g/L, and nitrogen source is that peptone concentration is 20mmol/L, and manganese ion concentration is 200 μm of ol/L.Pass through range analysis, arrangement
Each factor is to Index Influence size out are as follows: and RC > RA > RD > RE > RB illustrates that nitrogen source type produces MnP to Hericium erinaceus and influences maximum,
Followed by carbon source kind, nitrogen concentration, followed by manganese ion concentration and carbon source concentration.
Table 2 first time orthogonal test is to the influence result for producing MnP enzyme
Fig. 1~5 are the tendency chart of first time orthogonal test, show 5 kinds of factors under different level to the feelings for producing MnP enzyme
Condition.Glucose is known most beneficial for producing enzyme, cornstarch is most disadvantageous in producing enzyme.Influence of the different carbon source kinds to MnP enzyme is produced
It is bigger, but the concentration of carbon source is not very big to the influence for producing MnP enzyme.Different nitrogen sources type is maximum to the influence for producing MnP enzyme,
Nitrogen source is out of condition when being ammonium sulfate and dregs of beans, and furthermore nitrogen concentration also has certain influence to MnP enzyme is produced, so nitrogen source is to influence
One key factor of MnP enzyme secretion.The higher the better for manganese ion concentration in trial stretch.
Influence of second of orthogonal test to MnP enzyme is produced
On the basis of first time orthogonal test, second of orthogonal test is carried out, with liquid amount (mL), G when F static gas wave refrigerator
The concentration (mL/L) of Tween 80, H pH value, I MgSO4 ﹒ 7H2The concentration (g/L) of O is factor (being shown in Table 3), designs L9 (34) just
Table is handed over to carry out second of orthogonal test.
Second of orthogonal test obtains the optimum condition that Hericium erinaceus produces MnP are as follows: Tween 80 concentration is 0.25mL/L, culture solution
PH value 4.5, MgSO4·7H2O concentration is 0.3g/L.
3 Hericium erinaceus of table produces second of orthogonal test factor level design of MnP
It is F1G1H2I1 that optimal case is most obtained in second of orthogonal test, is shown in Table 4.Liquid amount is when cultivating
100mL, Tween 80 concentration are 0.25mL/L, and pH 4.5, MgSO47H2O concentration is 0.3g/L.Pass through range analysis, arrangement
Primary and secondary sequence of each factor to Index Influence size out are as follows: RF > RH > RG > RI, i.e. liquid amount and pH value influence maximum to producing enzyme,
Followed by Tween 80 concentration and MgSO47H2O concentration.
Second of the orthogonal test of table 4 is to the influence result for producing MnP enzyme
Fig. 6~9 are the tendency chart of second of orthogonal test, show 4 kinds of factors under different level to the feelings for producing MnP enzyme
Condition.The number of liquid amount had both reflected nutriment, also reflected opposite dissolved oxygen amount, it is seen that requirement of the Hericium erinaceus to dissolved oxygen amount is very
Greatly.The Tween 80 of more high concentration is unfavorable for producing MnP enzyme, and the concentration of pH value is advisable for 4.5, Mg2+Concentration should not be too high.
Influence of 5 different rotating speeds of table to MnP is produced
| Revolving speed/r/min | 100 | 150 | 200 | 250 | 300 |
| Enzyme activity U/L | 233.6 | 275.8 | 298.7 | 208.4 | 184.6 |
2 B of A, 1 C, 2 D, 3 E, 4 F, 1 G, 1 H, 2 I, 1 culture medium condition, the work of MnP are combined using optimum optimization
Property generated after 3d when static, and generated afterwards for 24 hours in shaken cultivation, revolving speed gradually rises to 200r/min therewith, and MnP enzyme produces
Measure highest；But when revolving speed is further increased to 300r/min, MnP enzyme activity is substantially reduced.It is thus determined that revolving speed is 200r/min.
Influence of 6 different temperatures of table to MnP is produced
| Temperature/DEG C | 20 | 24 | 28 | 32 | 36 |
| Enzyme activity U/L | 165.3 | 228.9 | 318.2 | 236.5 | 143.4 |
2 B of A, 1 C, 2 D, 3 E, 4 F, 1 G, 1 H, 2 I, 1 culture medium condition is combined using optimum optimization, in 5 kinds of temperature
Lower shaken cultivation 200r/min is spent, 7d measures enzyme activity, and when temperature is lower than 32 DEG C, overall producing enzyme effect is preferable, most suitable producing enzyme
Temperature is 28 DEG C, and enzyme activity reaches 318.2U/L at such a temperature；But when temperature is more than 32 DEG C, yield of enzyme declines rapidly, and 36
DEG C when, enzyme activity drops to 143.4U/L, and Hericium erinaceus is very high in the requirement of temperature, and strong high temperature inhibits to produce MnP enzyme, it is thus determined that
Optimum fermentation temp is 28 DEG C.
The different mineral element concentration of table 7 produce the influence of MnP
| Mineral element concentration/mL/L | 1 | 5 | 10 | 15 | 20 |
| Enzyme activity U/L | 360.5 | 362.4 | 364.5 | 381.6 | 368.3 |
2 B of A, 1 C, 2 D, 3 E, 4 F, 1 G, 1 H, 2 I, 1 culture medium condition is combined using optimum optimization, at 28 DEG C
The producing enzyme of lower shaken cultivation 200r/min, 7d are the result is that mineral element solution additional amount is 15mL/L, it is seen that mineral element
Concentration variation on produce MnP enzyme efficiency influence and it is little.
3, Optimum Experiment
It is final to obtain Hericium erinaceus production MnP most using the culture medium prescription and fermentation condition progress enzymatic production after optimization
Good condition are as follows: glucose concentration of medium 5g/L, peptone concentration 20mmol/L, manganese ion concentration are 200 μm of ol/L, spit
Warm 80 concentration are 0.25mL/L, MgSO4·7H2O concentration is 0.3g/L, mineral element solution additional amount is 15mL/L, culture solution pH
Value 4.5；28 DEG C of cultivation temperature；Liquid amount is 100mL under 200rpm shake flask culture conditions.Eventually by 3 groups of vibration-optimised cultures
Test, in 7d or so highest manganese peroxidase enzyme activity up to 400.6U/L, about 10.4 times before optimization.
4, fermentor is tested
Using 5L fermentor, liquid amount 3L, inoculum concentration 10%, periodically takes fermentation liquid 3mL daily by 28 DEG C of fermentation temperature,
5000r/min, 4 DEG C of centrifugation 5min take supernatant to measure manganese peroxidase enzyme activity.
The variation of 8 Hericium erinaceus fermentation process manganese peroxidase enzyme activity of table
| Fermentation time/d | 0 | 1 | 3 | 5 | 7 | 9 | 11 |
| Enzyme activity U/L | 0.015 | 162.6 | 385.5 | 426.7 | 531.8 | 464.6 | 401.2 |
With the extension of fermentation time, manganese peroxidase enzyme activity is also gradually increased, when fermentation proceeds to 7d,
Enzyme activity reaches maximum, and Figure 10 is that optimization culture Hericium erinaceus produces MnP enzyme curve graph, is tested using 5L fermentor, highest manganese peroxide
For compound enzyme activity up to 531.8U/L, highest enzyme activity is about 1.37 times under the conditions of laboratory shake flask；With fermentation time
It further increases, producing enzyme vigor is then begun to decline.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Claims (2)
1. a kind of method of whiterot fungi secretion manganese peroxidase, which is characterized in that
(1) solid medium PDA, including 180~220g of potato, 18~22g of glucose, 18~22g of agar powder, albumen are prepared
3~8g of peptone, distilled water are settled to 1000mL, and pH value is natural；
(2) by activated whiterot fungi-Hericium erinaceus on PDA plateHericium erinaceumStrain be made side length 8~
10mm fungus block accesses obtained fungus block in low nitrogen asparagine-succinic acid culture medium；
The low nitrogen asparagine-succinic acid culture medium includes that concentration of glucose is 5g/L, peptone concentration 20mmol/L, manganese
Ion concentration is 200 μm of ol/L, and Tween 80 concentration is 0.25mL/L, MgSO4﹒ 7H2O concentration is 0.3g/L, mineral element solution
Additional amount is 15mL/L, medium pH value 4.5；
Wherein, the mineral element solution of every 100 mL includes ironic citrate hydrate 0.0087g, ZnSO4·7H2O 0.01g,
MnSO4·H2O 0.045g, CoCl2·6H2O 0.01g, CuSO4·5H2O 0.001g；
It (3) is 100mL bottling, 8 fungus blocks of every bottle of inoculation, then in revolving speed 200r/min, 28 DEG C of shaking tables of temperature according to liquid amount
Middle constant-temperature shaking culture 7d measures manganese peroxidase enzyme activity；
(4) fermentor is tested, using 5L fermentor, liquid amount 3L, and inoculum concentration 10%, 28 DEG C of fermentation temperature, when fermentation proceeds to
When 7d, enzyme activity reaches maximum.
2. the method for whiterot fungi secretion manganese peroxidase according to claim 1, which is characterized in that described (1) is prepared
Solid medium PDA, including potato 200g, glucose 20g, agar powder 20g, peptone 5g, distilled water are settled to 1000mL,
PH value is natural.
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| CN106635843B (en) * | 2017-02-08 | 2019-09-27 | 金华职业技术学院 | Fermentation medium, mushroom bran fermenting agent and fermentation process |
| CN106906225B (en) * | 2017-02-23 | 2021-01-08 | 安庆师范大学 | Manganese peroxidase gene for degrading lignin and obtaining method thereof |
| CN111172126A (en) * | 2020-01-16 | 2020-05-19 | 黑龙江八一农垦大学 | Method for increasing yield of trametes hirsuta lignin peroxidase |
| CN111808831B (en) * | 2020-07-13 | 2022-09-02 | 浙江康星生物科技有限公司 | Preparation method of recombinant manganese peroxidase and application of recombinant manganese peroxidase in degradation of Chinese herbal medicine lignin |
| CN114908024B (en) * | 2022-06-23 | 2023-05-19 | 合肥工业大学 | Method for promoting white rot fungi to directionally produce enzyme to degrade plastic pollutants |
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