CN103146803A - Novel method for coupled enzymatic reaction-based high-flux detection of ethanol and C3-C6 n-alkanol production capacity of microbes - Google Patents
Novel method for coupled enzymatic reaction-based high-flux detection of ethanol and C3-C6 n-alkanol production capacity of microbes Download PDFInfo
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Abstract
The invention relates to a novel method of coupled enzymatic reaction-based high-flux detection of ethanol and C3-C6 n-alkanol production capacity of microbes. The novel method is characterized in that alcohol oxidase and catalase are coupled and the double-enzyme conjugate and ethanol or C3-C6 n-alkanol undergo a reaction in a corresponding buffer solution to produce red quinone imide; the double-enzyme coupling reaction is carried out in a 96-hole enzyme label plate and a light absorption value at the wavelength of 500nm is read by a microporous read device; a light absorption value at the wavelength of 500nm and a concentration of the reaction product of ethanol, C3-C6 n-alkanol and the double-enzyme conjugate are linearly dependent; according to a standard curve, concentration calculation is realized; and through the microporous read device and the 96-hole enzyme label plate, high-flux detection is realized.
Description
Technical field
The invention belongs to industrial microorganism screening and exploitation fields, particularly a kind of detection method that checks microorganisms producing ethanol and C3-C6 alphanol based on the high-throughput of two enzyme linked reactions.
Technical background
More than 200 year of past, the energy system that is based upon the fossil oil such as coal, oil, Sweet natural gas basis has greatly promoted the development of human society.Yet in recent years; excessive exploitation and consumption due to oil, Sweet natural gas, coal equal energy source; the world has faced serious energy dilemma; and International Crude Oil does not rise; the petroleum demand amount is increased day by day, since China in 1993 becomes petroleum products net importer first, annual crude oil import 21 in 2010; 8,450,000 tons, so the key that searching is cleaned, cheap, reproducible substitute energy becomes the future source of energy strategy.Alcohol fuel and fuel butanols are considered to replace the fossil oils such as oil, coal
The ideal chose of high pollution fossil oil as an alternative, biofuel are subject to already countries in the world and pay attention to.In recent years, the country such as the U.S., Brazil, Korea S, Indonesia and European Union have all put into effect the universal energy development strategy of biological support fuel in succession.European Union plans the year two thousand twenty and substitutes 20% fossil oil with biofuel." the national biofuel action plan " of the U.S. also proposes, and the year two thousand twenty biofuel will account for 25% of its energy aggregate consumption, and the year two thousand fifty reaches 50%.Alcohol fuel, fuel butanols are the recyclable fuels that is obtained through a series of physics, biology, chemical change process by Biological resources, have the advantages such as calorific value is high, performance good, easy to use.Propyl alcohol, amylalcohol and hexanol are all good industrial chemicals, are widely used in production and synthetic diet additive and the spices etc. of coating, printing-ink, cosmetics of everyday use, medicine and pesticide intermediate.The core that realizes biofuel production is the microbial strains of excellent.
In microbial metabolism, ethanol is released in substratum as product, in order to verify the producing and ethanol ability of microorganism, needs to measure the ethanol content in substratum.The common method of alcohol determining mainly comprises hydrometer method, colorimetry, vapor-phase chromatography and liquid phase chromatography at present.Hydrometer method is easy and simple to handle, can be used for the high-content alcohol determining, but to the alcohol determining specificity, but in microbial metabolism, the ethanol content of generation is difficult to reach the minimum that hydrometer method detects; Colorimetry as potassium bichromate colorimetry and iodimetry,iodometry, develops the color slow and unstable, disturbed by many factors, operates comparatively loaded down with trivial details; Chromatography can detect micro ethanol, and precision and accuracy are very high, but instrument is expensive, operation and maintenance is loaded down with trivial details.In addition, also have the report of the ethanol analysis methods such as infrared spectra, fluorescence and laser Raman spectroscopy, but still be in the laboratory study stage.Utilize in addition alcoholdehydrogenase can detect ethanol content, but alcoholdehydrogenase need to depend on NDA
+But, NDA
+Quite expensive, and need to go out to measure light absorption value at 340nm, therefore just need UV-detector, increased the expense that detects.Therefore, seeking easy, quick, accurate and widely used ethanol analysis method extremely payes attention to.
The screening method of producing and ethanol bacterial strain is more single at present, and report is arranged, and screens according to the color reaction of TTC (2,3,5-triphenyltetrazolium chloride).TTC is a kind of developer, the redox pigment, and the colourless solution that becomes soluble in water, but namely generate red after reduction and water-fast San Ben Ji Jia Za (TPF), TPF is more stable, can be by airborne oxygen autoxidation.Color reaction can be occured by the dehydrogenase oxidoreductase in microbial metabolism in TTC, has the height of desaturase or dehydrogenase activity but TTC can only illustrate bacterial strain, can not illustrate whether can produce ethanol.
In addition, ethanol and C3-C6 alphanol be available liquid chromatography and Gas chromatography detection all, but these two kinds of detection method determinations too bother, and the instrument price comparison is expensive, complex operation, this shows that present screening method and ethanol and C3-C6 alphanol detection method all are not suitable for the bacterial strain that high flux screening produces various alcohol, seek a kind of easy, various alphanols of method detection fast and accurately, significant for the suitable bacterial strain production ethanol of high flux screening and senior alphanol.
Summary of the invention
[purpose of the present invention]
The object of the present invention is to provide a kind of novel method of high throughput testing microorganisms producing ethanol and the C3-C6 alphanol based on two enzyme linked reactions.Method by alcohol oxidase and catalase coupling is measured ethanol and C3-C6 alphanol, high flux screening target microorganism.Compare with traditional microbe to screen method, low based on the high-throughput screening method cost of two enzyme linked reactions, efficient is high, purpose is stronger, can be from the good bacterial strain of occurring in nature screenability widely.
[thinking of the present invention]
Utilize the method for alcohol oxidase and catalase coupling, measure ethanol and C3-C6 alphanol content.Under the oxygen existence condition, alcohol is formed aldehyde and hydrogen peroxide by the alcohol oxidase oxidation, and the hydrogen peroxide of formation and 4-quinizine and phenol form a kind of red material quinonimine under the catalase effect.Quinonimine is a kind of red material, at the 500nm place, the characteristic absorbance value is arranged.Microbial metabolism produces the Organic Alcohol of ethanol and other C3-C6, reads the plate device by 96 hole enzyme plates and micropore, utilizes two enzyme coupling method analyzing and testing meta-bolitess, and the bacterial strain of the various alcohol of screening high yield is for fundamental research provides valuable microorganism resource.
[technological line of the present invention]
Technological line of the present invention is as follows:
(1) utilize substratum that screening sample is carried out enrichment culture 48h;
(2) mixed bacterium of enrichment culture is utilized sterilized water dilute (obtaining single bacterium colony), be applied to solid medium, cultivate 48h;
(3) the single bacterium colony that grows on solid medium is transferred in the 96 deep hole culture plates that contain liquid nutrient medium, single bacterium colony of each hole inoculation is built with silica gel, cultivates 48h;
(4) utilize refrigerated centrifuge to carry out 96 deep hole culture plates centrifugal, make bacterial sediment;
(5) get supernatant liquor in 96 deep hole culture plates and alcohol oxidase and catalase and react in 96 hole enzyme plates, 30 ℃ of insulation 30min;
(6) will react the 96 hole enzyme plates that finish and utilize micropore to read the light absorption value that the plate device is determined at 500nm place, and with the light absorption value measured with utilize the typical curve of alcohol oxidase and Catalase determination alcohol to compare, find pure concentration;
(7) will measure producing and ethanol or the high production bacterial strain of senior alphanol concentration is preserved.
[advantage of the present invention]
Advantage of the present invention is:
(1) efficient is high, and alcohol oxidase and catalase coupling can rapid detection ethanol or the content of senior alphanol.
(2) flux is high, and 96 deep hole culture plates and 96 hole enzyme plates are also read the plate device in conjunction with micropore and used together, make the screening of purpose bacterial strain wider.
(3) cost is low, can obtain at lower cost a large amount of bacterial classifications.
(4) can obtain more bacterial strains, for fundamental research provides microorganism resource.
Concrete enforcement
Be below the embodiment that enumerates, so that understand better the present invention.
Utilize the typical curve of the method mensuration ethanol of alcohol oxidase and catalase coupling.In Buffer I (200 μ L), each substances content is: phenol: 6mmol/ml, EDTA:75mg/L, phosphate buffered saline buffer: pH=7.5,0.1mol/L.In BufferII (100 μ L), each substances content is: alcohol oxidase: 3000u/L, catalase: 600u/L, 4-quinizine: 3.5m mol/L, phosphate buffered saline buffer 0.1mol/L pH=7.5.Preparation standard alcohol concn is respectively 0,0.1,0.5,1.0,1.5,2.0,2.5g/L.Step: first add Buffer I solution 200 μ L in 96 hole enzyme plates, the ethanol 10 μ L that add again different concns, add at last BufferII solution 100 μ L, process used is all carried out on ice, seals 30 ℃ of insulation 30min after mixing with sealer, under 500nm, measure light absorption value, set up the typical curve of OD500nm light absorption value and alcohol concn, R
2=0.999 (accompanying drawing 1) illustrates that the absorption value at alcohol concn and 500nm place is remarkable linear relationship.
Take hot spring soil sample 10g, utilize substratum at 60 ℃, carry out enrichment culture 24h under the 200rpm condition; Flora dilution 10 with enrichment culture
5, 10
6, 10
7Doubly, be applied on solid medium 60 ℃ of static cultivation 24h.The 96 deep hole culture plates that are 3mL with every pore volume add the substratum of 1mL, and single colony inoculation are arrived wherein 60 ℃ of static cultivation 24h.Utilize the Eppendorf refrigerated centrifuge with 96 deep hole culture plates at 4000rpm, centrifugal 30min under 4 ℃ of conditions.
First add Buffer I solution 200 μ L in 96 hole enzyme plates, then add supernatant liquor 10 μ L, add at last Buffer II solution 100 μ L, process used is all carried out on ice, seals 30 ℃ of insulation 30min after mixing with sealer, under 500nm, measure light absorption value.Calculate 96 strain bacterium ethanol productions according to the ethanol typical curve, obtain a highest bacterial strain of strain ethanol production.
The inoculation that above-mentioned screening is obtained at 200rpm, is cultivated 24h under 60 ℃ of conditions in substratum; Get the 10mL substratum, the centrifugal 5min of 10000rpm, utilize respectively method and the Syrups by HPLC alcohol concn of alcohol oxidase and catalase coupling, obtain concentration and be respectively 2.32g/L and 2.39g/L, two data are substantially identical, in limit of error, illustrate that the method mensuration ethanol of two enzyme couplings is reliable.
Getting pure propyl alcohol is mixed with concentration and is respectively 0.8,1.6,3.2,4.8,6.4,8.0g/L.First Buffer I solution 200 μ L in 96 hole enzyme plates, the n-propyl alcohol 10 μ L that add again different concns, add at last BufferII solution 100 μ L, process used is all carried out on ice, seal with sealer after mixing, 30 ℃ of insulation 30min measure light absorption value under 500nm, set up the typical curve (accompanying drawing 2) of OD500nm light absorption value and n-propyl alcohol concentration, R
2=0.999, illustrate within the specific limits, the absorption value at n-propyl alcohol concentration and 500nm place is remarkable linear relationship.
Embodiment 5
Getting pure butanols is mixed with concentration and is respectively 0,1.6,3.2,4.8,6.4,8.1g/L.First Buffer I solution 200 μ L in 96 hole enzyme plates, the propyl carbinol 10 μ L that add again different concns, add at last BufferII solution 100 μ L, process used is all carried out on ice, seal with sealer after mixing, 30 ℃ of insulation 30min measure light absorption value under 500nm, set up the typical curve (accompanying drawing 3) of OD500nm light absorption value and propyl carbinol concentration, R
2=0.998, illustrate within the specific limits, the absorption value at propyl carbinol concentration and 500nm place is remarkable linear relationship.
Getting pure amylalcohol is mixed with concentration and is respectively 40.0,48.5,65.0,70.0,81.0g/L.First Buffer I solution 200 μ L in 96 hole enzyme plates, the Pentyl alcohol 10 μ L that add again different concns, add at last BufferII solution 100 μ L, process used is all carried out on ice, seal with sealer after mixing, 30 ℃ of insulation 30min measure light absorption value under 500nm, set up the typical curve (accompanying drawing 4) of OD500nm light absorption value and Pentyl alcohol concentration, R
2=0.981, illustrate within the specific limits, the absorption value at Pentyl alcohol concentration and 500nm place is remarkable linear relationship.
Embodiment 7
Getting pure hexanol is mixed with concentration and is respectively 40.0,48.5,65.0,70.0,81.0g/L.First Buffer I solution 200 μ L in 96 hole enzyme plates, the n-hexyl alcohol 10 μ L that add again different concns, add at last Buffer II solution 100 μ L, process used is all carried out on ice, seal with sealer after mixing, 30 ℃ of insulation 30min measure light absorption value under 500nm, set up the typical curve (accompanying drawing 5) of OD500nm light absorption value and n-hexyl alcohol concentration, R
2=0.984, illustrate within the specific limits, the absorption value at n-hexyl alcohol concentration and 500mn place is linear.
Description of drawings
Accompanying drawing 1: alcohol concn and the typical curve of two enzyme linked reaction products in 500nm place's light absorption value foundation.
Accompanying drawing 2: n-propyl alcohol concentration and the typical curve of two enzyme linked reaction products in 500nm place's light absorption value foundation.
Accompanying drawing 3: propyl carbinol concentration and the typical curve of two enzyme linked reaction products in 500nm place's light absorption value foundation.
Accompanying drawing 4: Pentyl alcohol concentration and the typical curve of two enzyme linked reaction products in 500nm place's light absorption value foundation.
Accompanying drawing 5: n-hexyl alcohol concentration and the typical curve of two enzyme linked reaction products in 500nm place's light absorption value foundation
Claims (10)
1. high throughput testing novel method of utilizing detection microorganisms producing ethanol and the C3-C6 alphanol of alcohol oxidase and peroxidase linked reaction, step is as follows:
(1) substratum enrichment flora is applied in solid medium after dilution, is inoculated into after single bacterium colony grows in 96 deep hole culture plates and cultivates;
(2) utilize refrigerated centrifuge centrifugal under suitable condition 96 deep hole culture plates, get its supernatant liquor and measure;
(3) utilize the method for two enzyme couplings, measure pure content in above-mentioned supernatant liquor in conjunction with Buffer I and Buffer II in 96 hole enzyme plates.
2. by the method described in claim 1, it is characterized in that: described 96 deep hole culture plates are that volume is 3mL, but add the substratum of 0.5-2.5mL in every hole.
3. by the method described in claim 1, it is characterized in that: described refrigerated centrifuge is direct centrifugal 96 deep hole culture plates.
4. by the method described in claim 1, it is characterized in that: described centrifugal condition is 4 ℃, the centrifugal 10-60min of 4000rpm.
5. by the method described in claim 1, it is characterized in that: can not contain thalline in described supernatant liquor.
6. by the method described in claim 1, it is characterized in that: described pair of enzyme is alcohol oxidase and catalase.
7. by the method described in claim 1, it is characterized in that: in described Buffer I, each substances content is: phenol: 1-10mmol/ml, EDTA:25-150mg/L, phosphate buffered saline buffer: pH7.5,0.05-0.5mol/L.
8. by the method described in claim 1, it is characterized in that: in described Buffer II, each substances content is: alcohol oxidase: 500-6000U/L, catalase: 100-1000U/L, 4-quinizine: 1-5mmol/L, phosphate buffered saline buffer 0.05-0.5mol/L, pH=7.5.
9. by the method described in claim 1, it is characterized in that: described 96 hole enzyme plates need to be placed on ice, react 5-60min after Buffer I and Buffer II and sample join wherein under 30 ℃.
10. by the method described in claim 1, it is characterized in that: described mensuration wavelength is 500nm.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0117032A1 (en) * | 1983-01-12 | 1984-08-29 | Alcoholism And Drug Addiction Research Foundation | Rapid analysis of ethanol in body fluids |
| JP2003169696A (en) * | 2001-12-05 | 2003-06-17 | Toyobo Co Ltd | Method of measurement for biocomponent and reagent composition used in the same |
| CN1446925A (en) * | 2003-03-24 | 2003-10-08 | 肖洪武 | Single stable colorimetric reagent of enzyme in liquid state and its application |
| CN101762543A (en) * | 2008-12-10 | 2010-06-30 | 苏州艾杰生物科技有限公司 | Reagent (kit) for diagnosing/determining alcohol and method for determining concentration of alcohol |
| CN101942498A (en) * | 2009-07-07 | 2011-01-12 | 河北农业大学 | Kit for rapidly detecting diabetes |
-
2011
- 2011-12-07 CN CN2011104329160A patent/CN103146803A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0117032A1 (en) * | 1983-01-12 | 1984-08-29 | Alcoholism And Drug Addiction Research Foundation | Rapid analysis of ethanol in body fluids |
| JP2003169696A (en) * | 2001-12-05 | 2003-06-17 | Toyobo Co Ltd | Method of measurement for biocomponent and reagent composition used in the same |
| CN1446925A (en) * | 2003-03-24 | 2003-10-08 | 肖洪武 | Single stable colorimetric reagent of enzyme in liquid state and its application |
| CN101762543A (en) * | 2008-12-10 | 2010-06-30 | 苏州艾杰生物科技有限公司 | Reagent (kit) for diagnosing/determining alcohol and method for determining concentration of alcohol |
| CN101942498A (en) * | 2009-07-07 | 2011-01-12 | 河北农业大学 | Kit for rapidly detecting diabetes |
Non-Patent Citations (1)
| Title |
|---|
| 黄秀东等: "酶法测定甲醇酵母发酵液中甲醇浓度", 《生物技术》, vol. 11, no. 2, 30 April 2001 (2001-04-30) * |
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Owner name: TIANJIN INSTITUTE OF INDUSTRIAL BIOTECHNOLOGY, CHI Free format text: FORMER OWNER: TIANJIN INSTITUTE OF INDUSTRIAL BIOTECHNOLOGY Effective date: 20140319 |
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Application publication date: 20130612 |