CN101936909B - Enzyme activity testing method for micro-biological degradation of benzene ring - Google Patents
Enzyme activity testing method for micro-biological degradation of benzene ring Download PDFInfo
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Abstract
The invention discloses an enzyme activity testing method for micro-biological degradation of a benzene ring. The testing method comprises the following steps of: 1, making a standard curve of relation between substrate concentration and absorbance, and finding out a linear fitting equation between the substrate concentration and the absorbance; 2, preparing substrate solution according to enzyme or bacterial suspension of which the activity is to be measured; 3, previously heating the substrate solution and the enzyme solution to the most proper reaction temperature respectively, mixing the substrate solution and the enzyme solution, performing catalytic reaction on the mixture under the most proper reaction condition, and detecting the variation in the substrate concentration after the reaction; 4, measuring benzene ring content of the reaction solution, namely diluting the reaction solution before and after the catalytic reaction to ensure that the absorbance of the reaction solution is within the range of the linear equation, measuring the absorbance at the maximum absorption wavelength, and calculating the substrate concentration before and after the reaction according to the linear equation; and 5, calculating the enzyme activity U for the micro-biological degradation of the benzene ring according to a formula (1) of U=delta C/M/delta t*103.
Description
Technical field
The present invention relates to the measuring technology that enzyme is lived in the microbe whole-cell catalytic process, be specially a kind of enzyme method of testing alive of microbial degradation phenyl ring.
Background technology
Contain benzene ring substance and in chemical industry, have application widely, the industrial waste water quantity that therefore forms is more, and benzene ring substance has certain toxic action to environment; To biological even carcinogenic teratogenesis arranged; Therefore, the degraded of benzene ring substance is the heat subject of research.Biodegradation is a degradation of pollutant method commonly used, and phenyl ring has certain biodegradability, and therefore, microbial degradation contains the phenyl ring material also has more research.
Contain in the degradation process of phenyl ring material, opening of phenyl ring is the important indicator of estimating the degraded situation.Therefore, estimate the degradation capability of a kind of microorganism to pollutant, investigating its biology enzyme is crucial to the degradation rate of phenyl ring, and the enzyme of the phenyl ring of just degrading is lived.In the existing enzyme research method alive, generally judge enzyme activity through investigating the chromophoric variation of certain side group, but different because of the chromophore of every kind of material, cause this class testing method not have good universality.In the scope of applicant's retrieval, relevant directly employing phenyl ring open loop method is measured enzyme document alive and is not appeared in the newspapers as yet.
Summary of the invention
To the live limitation of method of testing of existing enzyme, the technical matters that quasi-solution of the present invention is determined is, a kind of enzyme of the microbial degradation phenyl ring method of testing of living is provided, and this method of testing has universality and substantivity well, and simple and easy to do.
The technical scheme that the present invention solve the technical problem is, the enzyme that the designs a kind of microbial degradation phenyl ring method of testing of living, and this method of testing may further comprise the steps:
1. make the typical curve that concerns between concentration of substrate and the absorbance: prepare a series of different substrate solutions that contain phenyl ring concentration; And on ultraviolet spectrophotometer, carry out full wavelength scanner; And then definite difference contains the maximum absorption wavelength of the substrate solution of phenyl ring concentration; Under its maximum absorption wavelength, measure the different absorbances that contain the substrate solution of phenyl ring concentration then; Draw the canonical plotting that concerns between concentration of substrate and the absorbance, find out that the linear fit degree reaches the concentration of substrate scope more than 0.999 between concentration of substrate and the absorbance, match obtains being used for calculating the linear equation of degradation process phenyl ring changes of contents; Said substrate is the material that contains phenyl ring;
2. preparation substrate solution: according to the enzyme of vigor to be measured or the catalytic condition of bacteria suspension, the preparation substrate solution;
3. catalytic reaction: substrate solution and enzyme liquid are heated to optimal reactive temperature separately in advance respectively, then substrate solution and enzyme liquid are mixed into reactant liquor, under optimum reaction conditions; Make it to take place catalytic reaction; And pick up counting, after the time through the catalytic reaction setting, take out reactant liquor; High-temperature inactivation detects concentration of substrate and changes rapidly;
4. the phenyl ring content in the assaying reaction liquid: the reactant liquor before and after the above-mentioned catalytic reaction is diluted corresponding multiple, make its absorbance in said linear equation scope; Reactant liquor after the dilution is the absorbance before and after the test reaction under maximum absorption wavelength, and calculates the concentration of substrate of reaction front and back according to said linear equation;
5. calculating the enzyme of microbial degradation phenyl ring lives: according to following formula (1), calculate the enzyme that obtains the microbial degradation phenyl ring and live:
Enzyme U=△ alive C/M/ △ t * 10
3(1)
(1) in the formula, △ C representes the change in concentration of substrate in the reactant liquor of catalytic reaction front and back, and unit is g/L; M representes the relative molecular weight of substrate, and unit is g/mol, and △ t representes the catalytic reaction time, and unit is min; 10
3Be the unit of each amount and the correcting value between the regulation enzyme unit alive.
It is catalytic media that survey method of the present invention adopts bacteria suspension, is substrate to contain the phenyl ring material, through the variation of phenyl ring absorbance in the catalytic degradation process, draws out corresponding typical curve, and the calculating through specific formulation can draw corresponding enzyme live data.Compared with prior art; The detection that method of testing of the present invention is lived for enzyme in the mass degradation process that contains phenyl ring has good universality; Be applicable to that majority contains the direct mensuration that enzyme is lived in the degradation process of benzene ring compound; And test process is simple, does not need valuable instrument and equipment, is very suitable for actual test and uses.
Description of drawings
Fig. 1 is the curve map that enzyme a kind of embodiment of method of testing alive (P-phthalic acid at concentration and absorbance relation) of microbial degradation phenyl ring of the present invention draws;
Fig. 2 is the curve map that the enzyme another kind of embodiment of method of testing alive (protocatechuic acid concentration and absorbance relation) of microbial degradation phenyl ring of the present invention draws.
Embodiment
Below in conjunction with embodiment method of testing of the present invention is described further:
The enzyme of the microbial degradation phenyl ring of the present invention's design method of testing (abbreviation method of testing) alive, this method of testing may further comprise the steps:
1. make and contain the typical curve that concerns between phenyl ring substrate (hereinafter to be referred as substrate) concentration and the absorbance: for preparing a series of variable concentrations contains the phenyl ring substance solution; And on ultraviolet spectrophotometer, carry out full wavelength scanner; And then the maximum absorption wavelength that contains the phenyl ring substance solution of definite variable concentrations; Under its maximum absorption wavelength, measure the absorbance that contains the phenyl ring substance solution of variable concentrations then; Draw the canonical plotting that concerns between concentration of substrate and the absorbance, find out that the linear fit degree reaches the concentration of substrate scope more than 0.999 between concentration of substrate and the absorbance, match obtains being used for calculating the linear equation of degradation process phenyl ring changes of contents;
2. preparation substrate solution: according to the enzyme of vigor to be measured or the catalytic condition of bacteria suspension, the preparation substrate solution; Described catalytic condition is meant that the sodium hydrogen phosphate of 3-4g/L potassium dihydrogen phosphate and 6-7g/L makes buffer solution, and maintenance system stable p H value=6.8-7.0 adds the substrate that contains phenyl ring at last, comprises terephthalic acid (TPA) or protocatechuic acid etc., is made into the solution of 1-2g/L.
Research shows that in said catalytic reaction liquid, the yeast extract powder that adds 1-3g/L is made organic nitrogen source, and can effectively urging into, microorganism lives thereby improve enzyme to the wear rate of carbon source.
3. catalytic reaction: substrate solution and enzyme liquid are heated to optimal reactive temperature (for example 37 ℃) separately in advance respectively, then substrate solution and enzyme liquid are mixed into reactant liquor, be meant temperature at optimum reaction conditions, the rotating speed of speed oscillation incubator etc.; For example: 37 ℃, under the 200r/min, make it to take place catalytic reaction; And pick up counting, through the time of catalytic reaction setting, for example behind the 30min; Take out reactant liquor, high-temperature inactivation detects the variation of concentration of substrate rapidly;
4. the phenyl ring content in the assaying reaction liquid: the reactant liquor before and after the above-mentioned catalytic reaction is diluted corresponding multiple, and for example 50-100 doubly makes its absorbance in said linear equation scope; Reactant liquor after the dilution is the absorbance before and after the test reaction under maximum absorption wavelength, and calculates the concentration of substrate of reaction front and back according to said linear equation.
5. calculating the enzyme of microbial degradation phenyl ring lives: in method of testing of the present invention, the definition that enzyme is lived is: 1ml enzyme liquid or bacteria suspension, in the time, the 1 μ mol that degrades contains the phenyl ring in the phenyl ring material at 1min, is defined as the enzyme U/ml of unit alive.According to following formula (1), calculate the enzyme that obtains the microbial degradation phenyl ring and live:
Enzyme U=△ alive C/M/ △ t * 10
3(1)
(1) in the formula, △ C representes the change in concentration of substrate in the reactant liquor of catalytic reaction front and back, and unit is g/L; M representes the relative molecular weight of substrate, and unit is g/mol, and △ t representes the catalytic reaction time, and unit is min; 10
3Be the unit of each amount and the correcting value between the regulation enzyme unit alive.
The present invention does not address part and is applicable to prior art.
Provide the specific embodiment of method of testing of the present invention below: specific embodiment only is for further explain the present invention, does not constitute the restriction to application claim of the present invention.
Embodiment 1
With the terephthalic acid (TPA) is sole carbon source, and the enzyme of bacillus F4 degraded terephthalic acid (TPA) is lived.
Adopt phosphate buffer (sodium hydrogen phosphate of 3g/L potassium dihydrogen phosphate and 7g/L) compound concentration to be respectively 1,2,5; 10,20,30; Terephthaldehyde's acid solution (standard solution) of 40mg/L carries out full wavelength scanner to terephthaldehyde's acid solution of being prepared on ultraviolet spectrophotometer, confirm that its maximum absorption wavelength is at 240nm; Then under the 240nm wavelength; Measure the terephthalic acid (TPA) solution absorbency of said variable concentrations, the curve map (referring to Fig. 1) that corresponding concentration-absorbance is drawn, the concentration range of finding out its linear fit degree (degree of fitting reaches 0.99986) up to standard is 1-40mg/L; Match obtains linear equation (2): Y=0.08342+0.07584 * X; Wherein X represents P-phthalic acid at concentration, and Y represents ultraviolet absorptivity, and linear equation (2) is used for calculating the variation of this routine degradation process phenyl ring content.
The catalytic condition of the bacteria suspension that enzyme to be measured is lived, the sodium hydrogen phosphate that needs to add 3g/L potassium dihydrogen phosphate and 7g/L is made buffer solution, and terephthaldehyde's acid solution of this buffer preparation 1g/L is adopted in maintenance system stable p H value=6.8.
With after the substrate solution sterilization, be heated to 37 ℃, the thalline after centrifugal is suspended in the solution after the sterilization, after mixing; At 37 ℃, oscillating reactions under the 200r/min, and pick up counting, through behind the 30min; Take out reactant liquor, rapid 95 ℃ of water-bath deactivation 5min, detect substrate and change:
With 50 times of the dilutions of the reactant liquor before and after the above-mentioned reaction, make it within the concentration range of said linear equation.Solution after the dilution is under the 240nm wavelength, and the absorbance before and after the test reaction is respectively 1.607 and 1.472.The concentration of calculating reaction front and back terephthalic acid (TPA) according to linear equation is respectively 1.004g/L and 0.915g/L; In the concentration value substitution enzyme formula alive (1) with the gained terephthalic acid (TPA), enzyme U=△ alive C/M/ △ t * 10
3, wherein, M is 166.13, and △ t is 30, and the enzyme work that promptly obtains this bacteria suspension is: U=0.018.
Embodiment 2
Adding dusty yeast is organic nitrogen source, and the enzyme of bacillus F4 degraded terephthalic acid (TPA) is lived.
The curve map that the mensuration of terephthalic acid (TPA) standard solution and corresponding concentration-absorbance is drawn is with embodiment 1 (referring to Fig. 1); Match obtains linear equation (2): Y=0.08342+0.07584 * X; Wherein, X represents P-phthalic acid at concentration, and Y represents ultraviolet absorptivity, and linear equation (2) can be used for calculating the variation of phenyl ring content in this routine degradation process.
In catalytic reaction liquid, adding dusty yeast makes organic nitrogen source and can urge effectively into that microorganism lives thereby improve enzyme to the wear rate of carbon source.For the raising degree of confirming that enzyme is lived; In reactant liquor, add the yeast extract powder of 1g/L, the sodium hydrogen phosphate that adds 3g/L potassium dihydrogen phosphate and 7g/L is simultaneously made buffer solution, maintenance system stable p H value=6.8; Add terephthalic acid (TPA) at last, be made into the solution of 1g/L.
Be heated to 37 ℃ with after the substrate solution sterilization, the thalline after centrifugal is suspended in the solution after the sterilization, after mixing, at 37 ℃, the 200r/min oscillating reactions, and pick up counting.Through behind the 30min, take out reactant liquor, rapid 95 ℃ of water-bath deactivation 5min detect substrate and change.
With 50 times of the dilutions of the reactant liquor before and after the above-mentioned reaction, make it within the concentration range of linear equation.Solution after dilution test reaction front and back absorbance under the 240nm wavelength is respectively 1.607 and 1.341.Calculate reaction front and back P-phthalic acid at concentration according to linear equation and be respectively 1.004g/L and 0.829g/L.
Substitution formula (1), enzyme U=△ alive C/M/ △ t10
3, (wherein M is 166.13, and △ t is 30) obtains the work of bacteria suspension enzyme and is: U=0.035.Find relatively that with result among the embodiment 1 adding dusty yeast can be with 1 times of the enzyme of bacteria suspension raising alive nearly.
Embodiment 3
With the protocatechuic acid is sole carbon source, and the enzyme of bacterium TJ degraded protocatechuic acid is lived.
Adopt phosphate buffer (sodium hydrogen phosphate of 4g/L potassium dihydrogen phosphate and 6g/L) compound concentration to be respectively 1,2,5; 10,20,30; The protocatechuic acid solution of 40mg/L, full wavelength scanner confirms that its maximum absorption wavelength is at 250nm; Under the 250nm wavelength, measure the variable concentrations solution absorbency, corresponding concentration-absorbance is made curve map (referring to Fig. 2), and the concentration range of finding out its linear fit degree (degree of fitting reaches 0.99971) up to standard is 1-40mg/L; Match obtains linear equation (3): Y=0.07613+0.07579 * X; Wherein X represents protocatechuic acid concentration, and Y represents ultraviolet absorptivity, the variation that linear equation (3) can be used for calculating phenyl ring content in the degradation process.
The catalytic condition of the bacteria suspension that enzyme to be measured is lived need add the sodium hydrogen phosphate of 4g/L potassium dihydrogen phosphate and 6g/L and make buffer solution, and maintenance system stable p H value is about 7, therefore, adopts the protocatechuic acid solution of buffer preparation 2g/L.
Be heated to 37 ℃ with after the substrate solution sterilization, the thalline after centrifugal is suspended in the solution after the sterilization, after mixing, at 37 ℃, the 200r/min oscillating reactions, and pick up counting.Through behind the 30min, take out reactant liquor, rapid 95 ℃ of water-bath deactivation 5min detect substrate and change.
With 100 times of the dilutions of the reactant liquor before and after the above-mentioned reaction, make it within the concentration range of linear equation.Solution after dilution test reaction front and back absorbance under the 250nm wavelength is respectively 1.592 and 1.491.Calculate reaction front and back P-phthalic acid at concentration according to linear equation and be respectively 2.000g/L and 1.867g/L.
Substitution formula (1), enzyme U=△ alive C/M/ △ t * 10
3, (wherein M is 154.12, and △ t is 30) obtains the work of bacteria suspension enzyme and is: U=0.029.
Embodiment 4
Adding dusty yeast is organic nitrogen source, and the enzyme of bacterium TJ degraded protocatechuic acid is lived.
The curve map that the mensuration of protocatechuic acid standard solution and corresponding concentration-absorbance is drawn is with embodiment 3, and match obtains linear equation (3): Y=0.07613+0.07579 * X, and wherein X represents protocatechuic acid concentration, and Y represents ultraviolet absorptivity.But linear equation (3) is used for calculating the variation of this routine degradation process phenyl ring content.
In catalytic reaction liquid, adding dusty yeast makes organic nitrogen source and can urge effectively into that microorganism lives thereby improve the enzyme enzyme to the wear rate of carbon source.For the raising degree of confirming that enzyme is lived; The yeast extract powder that in reactant liquor, adds 3g/L; The sodium hydrogen phosphate that adds 4g/L potassium dihydrogen phosphate and 6g/L is simultaneously made buffer solution, and maintenance system stable p H value is about 7, adds the solution that protocatechuic acid is made into 2g/L at last.
Be heated to 37 ℃ with after the substrate solution sterilization, the thalline after centrifugal is suspended in the solution after the sterilization, after mixing, at 37 ℃, the 200r/min oscillating reactions, and pick up counting.Through behind the 30min, take out reactant liquor, rapid 95 ℃ of water-bath deactivation 5min detect substrate and change.
With 100 times of the dilutions of the reactant liquor before and after the above-mentioned reaction, make it within the concentration range of linear equation.Solution after dilution test reaction front and back absorbance under the 250nm wavelength is respectively 1.592 and 1.388.Calculate reaction front and back P-phthalic acid at concentration according to linear equation and be respectively 2.000g/L and 1.731g/L.
Substitution formula (1), enzyme U=△ alive C/M/ △ t10
3, (wherein M is 154.12, and △ t is 30) obtains the work of bacteria suspension enzyme and is: U=0.058.Find relatively that with result among the embodiment 3 adding dusty yeast can live raising more than 1 times with the enzyme of bacteria suspension.
Adding dusty yeast is organic nitrogen source, and the enzyme of bacterium TJ degraded protocatechuic acid is lived.
The curve map that the mensuration of protocatechuic acid standard solution and corresponding concentration-absorbance is drawn is with embodiment 3, and match obtains linear equation (3): Y=0.07613+0.07579 * X, and wherein X represents protocatechuic acid concentration, and Y represents ultraviolet absorptivity.But linear equation (3) is used for calculating the variation of this routine degradation process phenyl ring content.
In catalytic reaction liquid, adding dusty yeast makes organic nitrogen source and can urge effectively into that microorganism lives thereby improve the enzyme enzyme to the wear rate of carbon source.For the raising degree of confirming that enzyme is lived; The yeast extract powder that in reactant liquor, adds 2g/L; The sodium hydrogen phosphate that adds 4g/L potassium dihydrogen phosphate and 6g/L is simultaneously made buffer solution, and maintenance system stable p H value is about 7, adds the solution that protocatechuic acid is made into 1.5g/L at last.
Be heated to 37 ℃ with after the substrate solution sterilization, the thalline after centrifugal is suspended in the solution after the sterilization, after mixing, at 37 ℃, the 200r/min oscillating reactions, and pick up counting.Through behind the 30min, take out reactant liquor, rapid 95 ℃ of water-bath deactivation 5min detect substrate and change.
With 75 times of the dilutions of the reactant liquor before and after the above-mentioned reaction, make it within the concentration range of linear equation.Solution after dilution test reaction front and back absorbance under the 250nm wavelength is respectively 1.592 and 1.324.Calculate reaction front and back P-phthalic acid at concentration according to linear equation and be respectively 1.500g/L and 1.235g/L.
Substitution formula (1), enzyme U=△ alive C/M/ △ t10
3, (wherein M is 154.12, and △ t is 30) obtains the work of bacteria suspension enzyme and is: U=0.057.Find relatively that with result among the embodiment 3 adding dusty yeast can be with 1 times of the enzyme of bacteria suspension raising alive nearly.
Claims (3)
1. the enzyme of the microbial degradation phenyl ring method of testing of living, this method of testing may further comprise the steps:
(1). make the typical curve that concerns between concentration of substrate and the absorbance: prepare a series of different substrate solutions that contain phenyl ring concentration; And on ultraviolet spectrophotometer, carry out full wavelength scanner; And then definite difference contains the maximum absorption wavelength of the substrate solution of phenyl ring concentration; Under its maximum absorption wavelength, measure the different absorbances that contain the substrate solution of phenyl ring concentration then; Draw the canonical plotting that concerns between concentration of substrate and the absorbance, find out that the linear fit degree reaches the concentration of substrate scope more than 0.999 between concentration of substrate and the absorbance, match obtains being used for calculating the linear equation of degradation process phenyl ring changes of contents; Said substrate is the material that contains phenyl ring;
(2). the preparation substrate solution: according to the enzyme of vigor to be measured or the catalytic condition of bacteria suspension, the preparation substrate solution;
(3). catalytic reaction: substrate solution and enzyme liquid are heated to optimal reactive temperature separately in advance respectively, then substrate solution and enzyme liquid are mixed into reactant liquor, under optimum reaction conditions; Make it to take place catalytic reaction; And pick up counting, after the time through the catalytic reaction setting, take out reactant liquor; High-temperature inactivation detects concentration of substrate and changes rapidly;
(4). the phenyl ring content in the assaying reaction liquid: the reactant liquor before and after the above-mentioned catalytic reaction is diluted corresponding multiple, make its absorbance in said linear equation scope; Reactant liquor after the dilution is the absorbance before and after the test reaction under maximum absorption wavelength, and calculates the concentration of substrate of reaction front and back according to said linear equation;
(5). the enzyme that calculates the microbial degradation phenyl ring is lived: according to following formula (1), calculate the enzyme that obtains the microbial degradation phenyl ring and live:
Enzyme U=Δ alive C/M/ Δ t * 10
3(1)
(1) in the formula, Δ C representes the change in concentration of substrate in the reactant liquor of catalytic reaction front and back, and unit is g/L; M representes the relative molecular weight of substrate, and unit is g/mol, and Δ t representes the catalytic reaction time, and unit is min; 10
3Be the unit of each amount and the correcting value between the regulation enzyme unit alive.
2. the enzyme of microbial degradation phenyl ring according to claim 1 method of testing alive is characterized in that described substrate is terephthalic acid (TPA) or protocatechuic acid; Described catalytic condition is meant that the sodium hydrogen phosphate of 3-4g/L potassium dihydrogen phosphate and 6-7g/L makes buffer solution, and maintenance system stable p H value=6.8-7.0 adds substrate at last, is made into the solution of 1-2g/L; Said optimal reactive temperature is 37 ℃; Said optimum reaction conditions is meant 37 ℃ of temperature, the rotating speed 200r/min of shaker incubator; The time that said catalytic reaction is set is 30min; The corresponding multiple of described dilution is 50-100 times.
3. the enzyme of the microbial degradation phenyl ring according to claim 1 and 2 method of testing of living is characterized in that in described catalytic reaction liquid, adding the yeast extract powder of 1-3g/L.
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| CN100592076C (en) * | 2008-04-15 | 2010-02-24 | 河海大学 | Preprocessing method for measuring desaturase activity of drinking water biological active carbon treatment process |
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| 刘玲等.白腐真菌降解硝基苯化合物的研究.《安徽农业科学》.2009,第37卷(第27期),13214-13215. * |
| 王松等.硝基苯微生物降解的优化条件研究.《安全与环境学报》.2008,第8 卷(第6 期),5-8. * |
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