CN108642122A - The method for detecting the degradation capability of lignin degrading bacterium - Google Patents
The method for detecting the degradation capability of lignin degrading bacterium Download PDFInfo
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- CN108642122A CN108642122A CN201810406473.XA CN201810406473A CN108642122A CN 108642122 A CN108642122 A CN 108642122A CN 201810406473 A CN201810406473 A CN 201810406473A CN 108642122 A CN108642122 A CN 108642122A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/21—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Pseudomonadaceae (F)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/924—Hydrolases (3) acting on glycosyl compounds (3.2)
- G01N2333/942—Hydrolases (3) acting on glycosyl compounds (3.2) acting on beta-1, 4-glucosidic bonds, e.g. cellulase
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Abstract
The invention discloses a kind of method of the degradation capability of detection lignin degrading bacterium, steps:Bacterium to be measured is scattered in sterile water, suspension is obtained；2) suspension applies sodium carboxymethylcellulose solid medium after sterilization, culture；3) a part of bacterium for taking step 2) to obtain is dyed with Congo red aqueous solution, is decolourized with NaCl aqueous solutions, has seen whether transparent circle generation；If there is transparent circle to illustrate that bacterium is known as degradation capability to wooden；4) the another bacterium part for taking the step 2) of transparent circle to obtain is inoculated into sodium carboxymethylcellulose fluid nutrient medium, cultivates to obtain crude enzyme liquid, crude enzyme liquid is taken to carry out the measurement of carboxymethylcelluloenzyme enzyme work and the measurement of filter paper enzyme activity respectively；It lives when carboxymethylcelluloenzyme enzyme and is higher than 2.5U/mL, meanwhile, filter paper enzyme activity is higher than 1.5U/mL, and the degradation capability of the lignin degrading bacterium of bacterium to be measured is strong.The method of the present invention is efficient, simple and direct, at low cost, can be used for extensive Testing and appraisal.
Description
Technical field
The invention belongs to field of bacteria, are related to a kind of method of the degradation capability of detection lignin degrading bacterium.
Background technology
China is as a large agricultural country, and there are about more than 700,000,000 tons of agricultural crop straws every year.Due to the reason of wherein lignin complexity
Change characteristic to be difficult to be utilized, be dropped or be incinerated eventually as solid waste, crop straw burning directly results in always suspension in air
The increase of grain substance, and the toxic gases such as sulfur dioxide, carbon monoxide are generated, harmful effect is generated to health.Meanwhile wood
Quality exists as the byproduct of wood hydrolysis industry and paper industry, due to lacking the processing method of environmental protection, becomes pollution
The harmful substance of environment.And in inside plants, since cellulose is protected by lignin, stable structure, it is extremely difficult to degrade.So far
Until, it still has more than after 95% lignin is directly discharged into rivers or concentration with " black liquor " and burns up, be seldom utilized effectively.This
A little waste liquids are discharged into rivers, it will the local surface water and groundwater of serious pollution threatens Drinking Water water head site, to environment and
Human health brings serious harm.Therefore in the degradation process of lignocellulosic, accelerate lignin fibre and be converted into humus
Just become key.The complexity and scrambling of lignocellulosic structure, determine the difficult of its biodegradation process.
The degradation of lignocellulosic is an oxidation process, needs the synergistic effect of a variety of enzymes, the synthesis of degradation enzyme need to be by hydrolyzing
Carbohydrate provides energy.It will produce many enzyme systems in the biodegradation process of lignocellulosic, main degrading enzyme has：
Lignin peroxidase, manganese peroxidase, laccase and cellulase etc..
Currently, there is an urgent need for a kind of methods of the degradation capability of detection lignin degrading bacterium.
Invention content
It is a kind of efficiently simple and direct, inexpensive and suitable extensive the purpose of the present invention is overcoming the deficiencies of the prior art and provide
The method of the degradation capability of the detection lignin degrading bacterium of application.
Technical scheme of the present invention is summarized as follows：
A method of the degradation capability of detection lignin degrading bacterium includes the following steps:
1) bacterium to be measured is scattered in sterile water, obtains suspension；
2) the sodium carboxymethylcellulose solid medium that the suspension that step 1) obtains applies after sterilization is taken to be trained in 37 DEG C
It supports 2-3 days；
3) part for taking the bacterium of step 2) acquisition is dyed with the Congo red aqueous solution of 1mg/mL, dye is discarded after 30min
Liquid is decolourized with the NaCl aqueous solutions of 1mol/L, discards destainer after 30min, seen whether transparent circle generation；If there is transparent circle
Illustrate that bacterium is known as degradation capability to wooden；
4) the another bacterium part for taking the step 2) of transparent circle to obtain is inoculated into sodium carboxymethylcellulose fluid nutrient medium,
37 DEG C, shaken cultivation under the conditions of 120r/min cultivates 2-3 days, obtains crude enzyme liquid, crude enzyme liquid is taken to carry out carboxymethylcelluloenzyme enzyme respectively
The measurement of measurement and filter paper enzyme activity living；It lives when carboxymethylcelluloenzyme enzyme and is higher than 2.5U/mL, meanwhile, filter paper enzyme activity is higher than 1.5U/
The degradation capability of mL, the lignin degrading bacterium of bacterium to be measured are strong.
Advantages of the present invention：
The method of the present invention is efficiently simple and direct, the time is short, at low cost, and the result of measurement is to various microbial inoculums in lignin degradation mistake
Effect in journey has direct illustration, can be used for extensive Testing and appraisal.
Description of the drawings
Fig. 1 is different strain CMC enzyme activity comparison diagrams.
Fig. 2 is different strain FPA enzyme activity comparison diagrams.
Fig. 3 is glucose standard curve figure.
Specific implementation mode
With reference to specific embodiment, the present invention is further illustrated.
Pseudomonad microbial inoculum (Pseudomonas) in June, 2016, Guangdong Province, converged agriculture in Guangzhou Guangzhou gardens from China
Medicine Co., Ltd purchases, http://sq26174706.china.herostart.com.
Trichoderma viride microbial inoculum (Trichoderma viride) in June, 2016, Shandong Province, Weifang City, Shandong was green from China
Gansu Province Bioisystech Co., Ltd purchases.http://shandonglvlong.cn.made-in-china.com.
Trichoderma harzianum microbial inoculum (Trichoderma harzianum) in June, 2016 is from China, Shandong Province, Weifang City, Shandong
Lv Long Bioisystech Co., Ltd purchases.http://shandonglvlong.cn.made-in-china.com.
Make detected bacterium with above-mentioned each bacterium, is only citing, the present invention is not imposed any restrictions.
Embodiment 1
A method of the degradation capability of detection lignin degrading bacterium includes the following steps:
1) bacterium to be measured is scattered in sterile water, obtains suspension；Bacterium to be measured is pseudomonad, trichoderma viride and Ha Ci wood
Mould；
2) the sodium carboxymethylcellulose solid medium that the suspension that step 1) obtains applies after sterilization is taken to be trained in 37 DEG C
It supports 2 days；
3) part for taking the bacterium of step 2) acquisition is dyed with the Congo red aqueous solution of 1mg/mL, dye is discarded after 30min
Liquid is decolourized with the NaCl aqueous solutions of 1mol/L, discards destainer after 30min, seen whether transparent circle generation；If there is transparent circle
Illustrate that bacterium is known as degradation capability to wooden；
4) the another bacterium part for taking the step 2) of transparent circle to obtain is inoculated into sodium carboxymethylcellulose fluid nutrient medium,
37 DEG C, shaken cultivation under the conditions of 120r/min cultivates 2 days, obtains crude enzyme liquid, crude enzyme liquid is taken to carry out carboxymethylcelluloenzyme enzyme work respectively
Measurement and filter paper enzyme activity measurement；It lives when carboxymethylcelluloenzyme enzyme and is higher than 2.5U/mL, meanwhile, filter paper enzyme activity is higher than 1.5U/
The degradation capability of mL, the lignin degrading bacterium of bacterium to be measured are strong.
After testing, it is 2.83U/mL, filter paper enzyme activity 1.92U/mL that pseudomonad carboxymethylcelluloenzyme enzyme, which is lived,；
It is 2.16U/mL, filter paper enzyme activity 0.98U/mL that trichoderma viride carboxymethylcelluloenzyme enzyme, which is lived,；
It is 1.97U/mL, filter paper enzyme activity 0.87U/mL that trichoderma harzianum carboxymethylcelluloenzyme enzyme, which is lived,.
See Fig. 1 and Fig. 2, detects that the degradation capability of pseudomonad lignin degrading bacterium is strong.
Bacterium to be measured is put into stalk and (stalk and carboxymethyl in the mixed fermentation liquid of sodium carboxymethylcellulose fluid nutrient medium
The ratio of sodium cellulosate fluid nutrient medium is 1g:50mL), straw degradative rate is detected after 14 days：
Pseudomonad straw degradative rate is 63%,
Trichoderma viride straw degradative rate is 52%,
Trichoderma harzianum straw degradative rate is 49%.
Embodiment 2
A method of the degradation capability of detection lignin degrading bacterium includes the following steps:
1) bacterium to be measured is scattered in sterile water, obtains suspension；Bacterium to be measured is pseudomonad, trichoderma viride and Ha Ci wood
Mould；
2) the sodium carboxymethylcellulose solid medium that the suspension that step 1) obtains applies after sterilization is taken to be trained in 37 DEG C
It supports 3 days；
3) part for taking the bacterium of step 2) acquisition is dyed with the Congo red aqueous solution of 1mg/mL, dye is discarded after 30min
Liquid is decolourized with the NaCl aqueous solutions of 1mol/L, discards destainer after 30min, seen whether transparent circle generation；If there is transparent circle
Illustrate that bacterium is known as degradation capability to wooden；
4) the another bacterium part for taking the step 2) of transparent circle to obtain is inoculated into sodium carboxymethylcellulose fluid nutrient medium,
37 DEG C, shaken cultivation under the conditions of 120r/min cultivates 3 days, obtains crude enzyme liquid, crude enzyme liquid is taken to carry out carboxymethylcelluloenzyme enzyme work respectively
Measurement and filter paper enzyme activity measurement；It lives when carboxymethylcelluloenzyme enzyme and is higher than 2.5U/mL, meanwhile, filter paper enzyme activity is higher than 1.5U/
The degradation capability of mL, the lignin degrading bacterium of bacterium to be measured are strong.
After testing, it is 2.79U/mL, filter paper enzyme activity 1.88U/mL that pseudomonad carboxymethylcelluloenzyme enzyme, which is lived,；
It is 2.13U/mL, filter paper enzyme activity 0.96U/mL that trichoderma viride carboxymethylcelluloenzyme enzyme, which is lived,；
It is 1.93U/mL, filter paper enzyme activity 0.80U/mL that trichoderma harzianum carboxymethylcelluloenzyme enzyme, which is lived,.
Detect that the degradation capability of pseudomonad lignin degrading bacterium is strong.
Carboxymethyl cellulose (CMC) enzyme activity determination：
Identical 25ml dryings tool plug scale test tube 4 is taken, 1 is used as blank tube, and 3 are waited for test tube, divides in each test tube
CMC-Na standard solution 1.5ml (the CMC-Na aqueous solutions of mass concentration 0.51%) is not measured not accurately, is put into 50 DEG C of water-baths pre-
Hot 5min after crude enzyme liquid is preheated to 50 DEG C, adds 0.5ml that will wait for test tube and blank tube simultaneously after fully shaking up in waiting in test tube
30min is reacted in 50 DEG C of water-baths, crude enzyme liquid 0.5ml is added in blank tube after reaction, by blank tube and waits for that test tube is same
When be added 3ml DNS reagents after, in boiling water bath react 5min, be quickly cooled down after taking-up, distilled water be used in combination to be settled to 25ml.In
It under 540nm wavelength, is returned to zero with blank tube, measures the absorbance of the solution after being reacted in test tube, it is true according to glucose standard curve
The content for determining reduced sugar calculates CMC enzyme activity.
Filter paper (FPA) enzyme activity determination：Take identical 25ml drying tool plug scale test tube 4,1 is used as blank tube, 3
Wait for test tube, it is accurate respectively in each test tube that filter paper 0.5g is added, it is put into 50 DEG C of water-baths and preheats 5min, crude enzyme liquid is preheated to
After 50 DEG C, add 0.5ml in waiting in test tube, will wait for that test tube and blank tube react 30min in 50 DEG C of water-baths simultaneously after fully shaking up,
Crude enzyme liquid 0.5ml is added in blank tube after reaction, by blank tube and after 3ml DNS reagents are added simultaneously in test tube, boiling
Water-bath 5min is quickly cooled down after taking-up, distilled water is used in combination to be settled to 25ml.It under 540nm wavelength, is returned to zero, is measured with blank tube
The absorbance of solution after being reacted in test tube, the content of reduced sugar is determined according to glucose standard curve, calculates FPA enzyme activity.
The measurement of glucose standard curve：6 identical 25ml dryings tool plug scale test tubes are taken, are separately added into 0；0.2；
0.4；0.6；0.8；The 1mg/mL glucose solutions of 1ml are used in combination distilled water polishing to 2ml, and it is molten that 3ml DNS are added after shaking up
Liquid after mixing well, is placed in boiling water bath and reacts 5min, is quickly cooled down after taking-up, and 25ml is settled to distilled water, measures
Light absorption value at 540nm wavelength.Using glucose content as abscissa, solution absorbance is ordinate, and it is bent to draw Glucose standards
Line.
Sodium carboxymethylcellulose (CMC-Na) solid culture based formulas：CMC-Na15g；NH4NO31g；Peptone 1g；
MgSO4·7H2O0.5g；KH2PO41g, agar 20g；PH is naturally, add water to 1L, 121 DEG C of sterilizing 20min.
Sodium carboxymethylcellulose (CMC-Na) Liquid Culture based formulas：CMC-Na15g；NH4NO31g；Peptone 1g；
MgSO4·7H2O0.5g；KH2PO41g adds water to 1L, and pH is naturally, 121 DEG C of sterilizing 20min.
The citrate buffer solution of 0.05mol/L p H5.0：
A liquid (0.1mol/L citric acid solutions)：Citric acid sample 21.014g accurately is weighed, after a small amount of distillation water dissolution,
1000ml volumetric flasks are moved into, fully shakes up after accurate constant volume, is saved backup in 4 DEG C of refrigerators.
B liquid (0.1mol/L sodium citrate solutions)：Sodium citrate sample 29.412g accurately is weighed, is filled with a small amount of distilled water
After dividing dissolving, 1000ml volumetric flasks are moved into, shakes up after accurate constant volume, is saved backup in 4 DEG C of refrigerators.
Above-mentioned A liquid 205ml, B liquid 295ml is taken, 1000ml volumetric flasks are moved into after mixing well, pH is adjusted after constant volume is
5.0, as 0.05mol/L, the citrate buffer solution of pH5.0 are saved backup in 4 DEG C of refrigerators.
The CMC-Na aqueous solutions of mass concentration 0.51%：
CMC-Na sample 0.51g accurately are weighed, appropriate citrate buffer solution is added, is placed in 50 DEG C of -70 DEG C of water-baths and adds
Heat after being sufficiently stirred dissolving, moves into 100ml volumetric flasks, is settled to 100ml with citrate buffer solution, mixes well, in 4 DEG C of ice
It is saved backup in case.
DNS reagents：
3,5-dinitrosalicylic acid 6.3g accurately are weighed, with distillation water dissolution, the NaOH that 262ml 2mol/L are added is molten
Itself and the 500ml solution for containing 185g sodium potassium tartrate tetrahydrates are sufficiently mixed by liquid, and 5g crystalline phenols and 5g anhydrous sodium sulfites is added,
Stirring moves into 1000ml volumetric flask constant volumes extremely after cooling, is stored in brown reagent bottle to abundant dissolving, is placed at room temperature for 7d stabilizations
It is spare afterwards.
The glucose solution (glucose standard) of 1mg/mL：
It is accurate to weigh 0.1g, it is dried to the glucose of weight under the conditions of 105 DEG C, after a small amount of distillation water dissolution, moves into 100ml
Volumetric flask constant volume fully shakes up, and is saved backup in 4 DEG C of refrigerators.
Claims (1)
1. a kind of method of the degradation capability of detection lignin degrading bacterium, includes the following steps:
1) bacterium to be measured is scattered in sterile water, obtains suspension；
2) the sodium carboxymethylcellulose solid medium that the suspension that step 1) obtains applies after sterilization is taken to cultivate 2-3 in 37 DEG C
It；
3) part for taking the bacterium of step 2) acquisition is dyed with the Congo red aqueous solution of 1mg/mL, dye liquor is discarded after 30min, used
The NaCl aqueous solutions of 1mol/L decolourize, and discard destainer after 30min, have seen whether transparent circle generation；If there is transparent circle explanation
Bacterium is known as degradation capability to wooden；
4) the another bacterium part for taking the step 2) of transparent circle to obtain is inoculated into sodium carboxymethylcellulose fluid nutrient medium, and 37
DEG C, shaken cultivation under the conditions of 120r/min cultivates 2-3 days, obtains crude enzyme liquid, crude enzyme liquid is taken to carry out carboxymethylcelluloenzyme enzyme work respectively
Measurement and filter paper enzyme activity measurement；It lives when carboxymethylcelluloenzyme enzyme and is higher than 2.5U/mL, meanwhile, filter paper enzyme activity is higher than 1.5U/
The degradation capability of mL, the lignin degrading bacterium of bacterium to be measured are strong.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111690539A (en) * | 2020-07-07 | 2020-09-22 | 安徽农业大学 | Screening and application of high-efficiency straw cellulose decomposition bacteria |
| CN114574370A (en) * | 2022-03-18 | 2022-06-03 | 贵州医科大学 | Screening method of biomass degrading strain, trichoderma and application |
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| CN102876589A (en) * | 2012-10-17 | 2013-01-16 | 贵州茅台酒厂（集团）习酒有限责任公司 | Strains with high cellulase activity as well as screening method and use method of strains |
| CN105255735A (en) * | 2015-10-16 | 2016-01-20 | 河源职业技术学院 | Cellulase producing strains as well as screening method and application thereof |
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| CN102876589A (en) * | 2012-10-17 | 2013-01-16 | 贵州茅台酒厂（集团）习酒有限责任公司 | Strains with high cellulase activity as well as screening method and use method of strains |
| CN105255735A (en) * | 2015-10-16 | 2016-01-20 | 河源职业技术学院 | Cellulase producing strains as well as screening method and application thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111690539A (en) * | 2020-07-07 | 2020-09-22 | 安徽农业大学 | Screening and application of high-efficiency straw cellulose decomposition bacteria |
| CN114574370A (en) * | 2022-03-18 | 2022-06-03 | 贵州医科大学 | Screening method of biomass degrading strain, trichoderma and application |
| CN114574370B (en) * | 2022-03-18 | 2023-09-15 | 贵州医科大学 | Screening method of degradation biomass strain, trichoderma and application |
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