CN105524891A - Method for improving activity of lignin-degrading enzyme generated by phanerochaete chrysosporium after enzyme activity decay - Google Patents
Method for improving activity of lignin-degrading enzyme generated by phanerochaete chrysosporium after enzyme activity decay Download PDFInfo
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Abstract
The invention discloses a method for improving the activity of lignin-degrading enzyme generated by phanerochaete chrysosporium after enzyme activity decay. The method comprises the following steps: fermenting the phanerochaete chrysosporium in a carbon-limiting culture medium, and adding glucose to a fermentation system when a carbon source is used up, so that the activity of the lignin-degrading enzyme generated by the phanerochaete chrysosporium is improved after decay. According to the method, the recovery of the lignin-degrading enzyme activity can be realized only by adding an appropriate amount of glucose to the culturing system, the conditions are easy to control, and the method is suitable for the stable production and practical degradation of the lignin-degrading enzyme generated by phanerochaete chrysosporium.
Description
Technical field
The present invention relates to biological technical field, after particularly relating to the decay of a kind of Phanerochaete chrysosporium lignin degradation enzyme activity, improve the method that enzyme is lived.
Background technology
White-rot fungi is a kind of high filamentous fungus, and in classification, great majority belong to Basidiomycetes, grow on trees or timber, gains the name because causing wooden white to rot.White rot fungus degrading xylogen, is because it can produce lignin-degrading enzymes, and is secreted into extracellular.The degraded of lignin-degrading enzymes to xylogen is the chain reaction process based on free radical, there is extremely strong oxidisability and substrate non-specific, this degradation mechanism makes lignin-degrading enzymes can not only lignin degrading, can also to degrade many xenobionticses in environment and persistence toxic organic pollutant (Barr & Aust, 1994; Cameronetal., 2000; Gaoetal., 2010).Therefore, white-rot fungi and lignin-degrading enzymes thereof have important using value in environment pollution control and biological restoration etc. thereof.
Phanerochaete chrysosporium (Phanerochaetechrysosporium) is type culture (Asgheretal., 2008 that white-rot fungi produces lignin-degrading enzymes and the research of degraded persistence organic pollutant; Singh & Chen, 2008; Gaoetal., 2010), belong to Aphyllophorales (Phyllophorales), Fu Geke (Corticiaceae) and aobvious lead fungi and belong to (Phanerochaete).Phanerochaete chrysosporium Ligninolytic Enzymes system mainly comprises lignin peroxidase (LiP, EC1.11.1.14), manganese peroxidase (MnP, EC1.11.1.13) and produces H
2o
2oxydase.Lignin peroxidase and manganese peroxidase are the multiple isozyme of glycosylated iron content, lignin peroxidase can catalysis to the oxidation of non-phenolic lignin pattern compound and aromatic pollution, manganese peroxidase can catalysis to the oxidation of the xylogen pattern compound of xylogen, lignin derivative and a lot of phenols.The synthesis of Phanerochaete chrysosporium lignin-degrading enzymes is subject to the complexity adjustment of multiple nutrients and environmental factors, lignin-degrading enzymes can be subject to carbon, nitrogen or sulphur nutrition restriction time excite generation, performance active (Faison & Kirk, 1985 under hyperoxic conditions; Dosoretzetal., 1990a; Micheletal., 1992; Singh & Chen, 2008).It is generally acknowledged that Phanerochaete chrysosporium does not produce polyfunctional peroxide enzyme (versatileperoxidase) and laccase.
Usually, the white-rot fungis such as Phanerochaete chrysosporium produce lignin-degrading enzymes in culturing process, enzyme work is after reaching maximum, start rapidly to decline, enzyme is lived and be there is serious wild effect, and this is that white-rot fungi is applied to actual contaminant degradation and lignin-degrading enzymes commercially produces one of faced key constraints (Singh & Chen2008).Lignin degradation enzyme activity decay reason, two aspects may be comprised: one be the oxydase such as white-rot fungi glyoxal oxidase produce be present in extracellular H
2o
2to lignin-degrading enzymes, there is detrimental effect; Two is that the extracellular protease produced in the secondary metabolism stage has hydrolytic action (Dosoretzetal., 1990b, c to lignin-degrading enzymes; Dassetal., 1995; Xiongetal., 2008).At present, utilize Phanerochaete chrysosporium to produce the research of lignin-degrading enzymes, mainly comprise following aspect (Ikehataetal., 2004; Singh & Chen, 2008): the nutritional condition (comprising suitable carbon source, nitrogenous source, trace element, dissolved oxygen and inductor etc.) that (1) ferments; (2) envrionment conditions (as temperature, pH, stirring and fixing condition etc.) of fermenting; (3) research of reactor fermentation scale-up.So far, produce in the research of lignin-degrading enzymes Phanerochaete chrysosporium, still do not see and effectively can maintain lignin-degrading enzymes high level vigor or after enzyme lives decay, again improve enzyme method alive, the stable generation of lignin-degrading enzymes is still white-rot fungi and the required Main Bottleneck problem solved of lignin-degrading enzymes practical application.
It is Ligninases from White Rot Fungi especially one of the basic training method that produces of lignin peroxidase that carbon restriction is cultivated.Therefore, the stable generation realizing lignin-degrading enzymes under carbon restriction culture condition has great importance.
Summary of the invention
An object of the present invention is to provide a kind of method that lignin degradation enzyme activity that Phanerochaete chrysosporium is produced again improves after decay.
Method provided by the invention, comprise the steps: to ferment in carbon restriction substratum Phanerochaete chrysosporium, add carbon source when carbon source in fermentation system exhausts, the lignin degradation enzyme activity realizing described Phanerochaete chrysosporium is produced again improves after decay.
In aforesaid method, described carbon source is glucose;
The described amount adding glucose is 2-5g/L for making the glucose concn in fermentation system.
In aforesaid method, described in add carbon source number of times be 2-3 time.
In aforesaid method, adding glucose when described carbon source in fermentation system exhausts is following 1) or 2):
1), in fermentation culture within the 3rd day, in fermentation system, add glucose, make the glucose concn in described fermentation system be 2g/L; And within the 5th day, in fermentation system, add glucose in fermentation culture, make the glucose concn in described fermentation system be 2g/L;
2), in fermentation culture within the 3rd day, in fermentation system, add glucose, make the glucose concn in described fermentation system be 5g/L; And within the 6th day, in fermentation system, add glucose in fermentation culture, make the glucose concn in described fermentation system be 5g/L.
In aforesaid method, described carbon restriction substratum for add in carbon confined liquid substratum by Immobilized culture carrier, and makes its piling height higher than liquid nutrient medium liquid level, is in non-submerged state, the substratum obtained; The concentration of described Immobilized culture carrier in described carbon restriction substratum is 1.6g/100mL;
Carbon restriction substratum described in every 1L by final concentration be 5.04g/L glucose, final concentration is 4.05g/L ammonium tartrate, final concentration is 2.0g/LKH
2pO
4, final concentration is 0.5g/LMgSO
4, final concentration is 0.1g/LCaCl
2, final concentration is 1mg/L vitamins B
1, final concentration is 1.5mM veratryl alcohol, pH4.4 and final concentration is 20mM Acetic acid-sodium acetate damping fluid and 70mL/L trace element solution composition;
Described trace element solution is 3g/LMgSO by final concentration
4, final concentration is 0.5g/LMnSO
4, final concentration is 1.0g/LNaCl, final concentration is 0.1g/LFeSO
47H
2o, final concentration are 0.1g/LCoCl
2, final concentration is 0.1g/LZnSO
47H
2o, final concentration are 0.1g/LCuSO
4, final concentration is 10mg/LAlK (SO
4)
212H
2o, final concentration are 10mg/LH
3bO
3, final concentration is 10mg/LNa
2moO
42H
2o, final concentration are 1.5g/L nitrilotriacetic acid(NTA) salt and water composition.
In aforesaid method, described fermentation obtains fermentation system for being accessed by Phanerochaete chrysosporium spore in described carbon restriction substratum, cultivates; Phanerochaete chrysosporium spore content in described fermentation system is 1.0 × 10
5-1.0 × 10
9individual spore/mL.
In aforesaid method, the temperature of described cultivation is 35-39 DEG C, and the rotating speed of described cultivation is 140-170rpm.
In aforesaid method, described lignin-degrading enzymes is lignin peroxidase or manganese peroxidase.
Above-mentioned method is also the scope of protection of the invention producing the application in lignin-degrading enzymes or raising lignin degradation enzyme activity.
Another object of the present invention is to provide a kind of method that Phanerochaete chrysosporium produces lignin-degrading enzymes.
Method provided by the invention, comprises the step of above-mentioned method.
Use of the present invention proves, the present invention (does not supplement pure oxygen) under air ambient, carry out carbon restriction to immobilization Phanerochaete chrysosporium to cultivate, fermentation produces lignin-degrading enzymes, after glucose exhausts, by adding the strategy of glucose to culture system, achieve lignin peroxidase and the again raising of manganese peroxidase enzyme activity after decay.This lignin-degrading enzymes vitality restoration technology, only in culture system, glucose need be added in right amount, the recovery that lignin-degrading enzymes enzyme is lived can be realized, condition controls simple, has important reference significance to the actual degraded application of the stably manufactured and white-rot fungi that realize Phanerochaete chrysosporium lignin-degrading enzymes.
Accompanying drawing explanation
Fig. 1 is the impact that glucose feed supplement produces at carbon restriction culturing process lignin peroxidase Phanerochaete chrysosporium.
Fig. 2 is the impact that glucose feed supplement produces at carbon restriction culturing process manganese peroxidase Phanerochaete chrysosporium.
Fig. 3 is the consumption that under glucose feed supplement effect, Phanerochaete chrysosporium limits culturing process glucose at carbon.
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
In following embodiment, Phanerochaete chrysosporium BKM-F-1767 spore is documented in as in Publication about Document: Tien, M., Kirk, T.K.LigninperoxidaseofPhanerochaetechrysosporium.Methods inEnzymology.1988,161:238-249.
Embodiment 1, Phanerochaete chrysosporium improve enzyme after the decay of lignin degradation enzyme activity and live under carbon restriction is cultivated
The Phanerochaete chrysosporium method that after the decay of lignin degradation enzyme activity, raising enzyme is lived under carbon restriction is cultivated is in Phanerochaete chrysosporium fermenting process, 2g/L glucose is added after carbon source exhausts, realize the rear enzyme that improves of Phanerochaete chrysosporium lignin degradation enzyme activity decay under carbon restriction is cultivated to live, specific as follows:
1, the preparation of carbon confined liquid substratum
Adopt carbon confined liquid substratum, substratum revises a little based on typical Tien and Kirk substratum (1988) to form, and culture medium C/N ratio (mol ratio) is 3.8, specifically composed as follows:
Carbon confined liquid substratum by final concentration be 5.04g/L (28mM) glucose, final concentration is 4.05g/L (NH
4 +44mM) ammonium tartrate, final concentration are 2.0g/LKH
2pO
4, final concentration is 0.5g/LMgSO
4, final concentration is 0.1g/LCaCl
2, final concentration is 1mg/L vitamins B
1, final concentration is 1.5mM veratryl alcohol, final concentration is 20mM (pH4.4) Acetic acid-sodium acetate damping fluid, 70mL/L trace element solution and deionized water composition; PH is 4.4.
Trace element solution is 3g/LMgSO by final concentration
4, final concentration is 0.5g/LMnSO
4, final concentration is 1.0g/LNaCl, final concentration is 0.1g/LFeSO
47H
2o, final concentration are 0.1g/LCoCl
2, final concentration is 0.1g/LZnSO
47H
2o, final concentration are 0.1g/LCuSO
4, final concentration is 10mg/LAlK (SO
4)
212H
2o, final concentration are 10mg/LH
3bO
3, final concentration is 10mg/LNa
2moO
42H
2o, final concentration are 1.5g/L nitrilotriacetic acid(NTA) salt and water composition.
2, maintain Phanerochaete chrysosporium and produce lignin-degrading enzymes enzyme stability alive
The polyurethane foamed blocks of 1.6g length of side 1.5cm is placed in the 250mL Erlenmeyer flask that 100mL carbon confined liquid substratum is housed, as Immobilized culture carrier, carrier is in non-submerged state, substratum and carrier sterilizing (115 DEG C, 30min), obtain carbon restriction substratum.
To grow in the Phanerochaete chrysosporium BKM-F-1767 spore aseptic access carbon restriction substratum on 30 DEG C of PDA flat boards (200g potato leach liquor/L, 20g glucose/L, 20g agar/L), after inoculation, spore concentration is about 1.0 × 10
5spore/mL, obtains culture system.
Be 37 DEG C by the culture system of above-mentioned access spore in air ambient (not being filled with pure oxygen in addition) temperature, rotating speed is that 160rpm (rotation radius 1.25cm) condition bottom fermentation is cultivated, in culturing process, make the glucose concn in culture system be 2g/L detecting after glucose exhausts that (fermentation culture the 3rd day) adds dextrose in sterile water to culture system, continue to cultivate; Again detect glucose exhaust after (fermentation culture the 5th day) again add dextrose in sterile water to culture system and make glucose concn in culture system be 2g/L, continue to cultivate.
Control group: do not add glucose to culture system.
Three groups of parallel laboratory tests are carried out in control experiment and glucose fed experiment all simultaneously, and result is the mean value of three groups of experiments.
Lignin peroxidase activity employing is that the method for substrate measures with veratryl alcohol, and every 1min is oxidized 1 μm of ol veratryl alcohol and becomes the enzyme amount needed for veratryl aldehyde to be defined as 1 enzyme activity unit.
Manganese peroxidase enzymic activity adopts with Mn
2+for the methods analyst of substrate, every 1min is oxidized 1 μm of olMn
2+required enzyme amount is defined as 1 enzyme activity unit.
Glucose concn adopts glucose analysis test kit (Shanghai Shensuo Youfu Medical Diagnosis Products Co., Ltd., product standard numbering YZB/ Shanghai 1527-40-2011) to measure.
Detect the generation of culturing process lignin peroxidase and manganese peroxidase, result respectively as depicted in figs. 1 and 2, the adding and consuming as shown in Figure 3 of culturing process glucose.Result shows, when not adding glucose, namely lignin peroxidase vigor starts after reaching maximum at the 3rd day to decline, and manganese peroxidase enzyme activity also starts to decline after the 3rd and 4 days reach maximum.By comparison comparatively, after glucose runs out for twice, in culture system, add 2g/L glucose for twice, after glucose fed, all significantly improve the vigor of lignin peroxidase and manganese peroxidase.
Embodiment 2, Phanerochaete chrysosporium improve enzyme after the decay of lignin degradation enzyme activity and live under carbon restriction is cultivated
The Phanerochaete chrysosporium method that after the decay of lignin degradation enzyme activity, raising enzyme is lived under carbon restriction is cultivated is in Phanerochaete chrysosporium fermenting process, 5g/L glucose is added after carbon source exhausts, realize the rear enzyme that improves of Phanerochaete chrysosporium lignin degradation enzyme activity decay under carbon restriction is cultivated to live, specific as follows:
1, the preparation of carbon confined liquid substratum: identical with embodiment 1;
2, maintain Phanerochaete chrysosporium and produce lignin-degrading enzymes enzyme stability alive
Substantially the same manner as Example 1, the step of glucose is added in different being only:
Be 37 DEG C by the culture system of above-mentioned access spore in air ambient (not being filled with pure oxygen in addition) temperature, rotating speed is that 160rpm condition bottom fermentation is cultivated, in culturing process, make the glucose concn in culture system be 5g/L detecting after glucose exhausts that (fermentation culture the 3rd day) adds dextrose in sterile water to culture system, continue to cultivate; Again detect glucose exhaust after (fermentation culture the 6th day) again add dextrose in sterile water to culture system and make glucose concn in culture system be 5g/L, continue to cultivate.
Detection method, with embodiment 1, detects the generation of culturing process lignin peroxidase and manganese peroxidase, result respectively as depicted in figs. 1 and 2, the adding and consuming as shown in Figure 3 of culturing process glucose.Result shows, when not adding glucose, namely lignin peroxidase vigor starts after reaching maximum at the 3rd day to decline, and manganese peroxidase enzyme activity also starts to decline after the 3rd and 4 days reach maximum.By comparison comparatively, after glucose consumption is most, 5g/L glucose is added in culture system, although lignin peroxidase vigor is subject to of short duration suppression (the 4th day) because carbon source concentration does not meet carbon restricted condition, is activated very soon and has recovered higher lignin peroxidase vigor (the 5th and 6 days); After manganese peroxidase enzyme activity then adds 5g/L glucose at twice, show raising in various degree (the 4th and 7 days).
Claims (9)
1. the method that again improves after decay of the lignin degradation enzyme activity that Phanerochaete chrysosporium is produced, comprise the steps: to ferment in carbon restriction substratum Phanerochaete chrysosporium, add carbon source when carbon source in fermentation system exhausts, the lignin degradation enzyme activity realizing described Phanerochaete chrysosporium is produced again improves after decay.
2. method according to claim 1, is characterized in that: described carbon source is glucose; The described amount adding glucose is 2-5g/L for making the glucose concn in fermentation system.
3. method according to claim 1 and 2, is characterized in that: described in add carbon source number of times be 2-3 time.
4. method according to claim 3, is characterized in that:
Adding glucose when described carbon source in fermentation system exhausts is following 1) or 2):
1), in fermentation culture within the 3rd day, in fermentation system, add glucose, make the glucose concn in described fermentation system be 2g/L; And within the 5th day, in fermentation system, add glucose in fermentation culture, make the glucose concn in described fermentation system be 2g/L;
2), in fermentation culture within the 3rd day, in fermentation system, add glucose, make the glucose concn in described fermentation system be 5g/L; And within the 6th day, in fermentation system, add glucose in fermentation culture, make the glucose concn in described fermentation system be 5g/L.
5., according to described method arbitrary in claim 1-4, it is characterized in that:
Described fermentation obtains fermentation system for being accessed by Phanerochaete chrysosporium spore in described carbon restriction substratum, cultivates; Phanerochaete chrysosporium spore content in described fermentation system is 1.0 × 10
5-1.0 × 10
9spore/mL.
6., according to described method arbitrary in claim 1-5, it is characterized in that:
The temperature of described cultivation is 35-39 DEG C, and the rotating speed of described cultivation is 140-170rpm.
7., according to described method arbitrary in claim 1-6, it is characterized in that: described lignin-degrading enzymes is lignin peroxidase and/or manganese peroxidase.
8. in claim 1-7, arbitrary described method is producing the application in lignin-degrading enzymes and/or raising lignin degradation enzyme activity.
9. Phanerochaete chrysosporium produces a method for lignin-degrading enzymes, comprises the step of arbitrary described method in claim 1-7.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106036007A (en) * | 2016-06-08 | 2016-10-26 | 莫竹承 | Biodegradation method for spartina alterniflora lignin |
| CN108383611A (en) * | 2018-04-19 | 2018-08-10 | 赵建平 | A kind of preparation method of organic fertilizer for flower |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108383611A (en) * | 2018-04-19 | 2018-08-10 | 赵建平 | A kind of preparation method of organic fertilizer for flower |
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