Novel method for coupled enzymatic reaction-based high-flux detection of ethanol and C3-C6 n-alkanol production capacity of microbes

A C3-C6, microbial technology, applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of cumbersome operation, expensive equipment, troublesome pre-treatment, etc., and achieve high throughput, low cost, and low cost. cost effect

Inactive Publication Date: 2013-06-12
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Claims
  • Application Information

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Problems solved by technology

[0006] In addition, ethanol and C3-C6 n-alkanols can be detected by liquid chromatography and gas chromatography, but the pretreatment of samples by these two detection methods is too troublesome, and the instrument price is relatively expensive, and the operation is cumbersome. This shows that the current screening method And ethanol and C3-C6 n-alkanol detection methods are not suitable for high-throughput screening of strains producing various alcohols, looking for a simple, fast and accurate method to detect various n-alkanols, for high-throughput screening of suitable strains It is of great significance to produce ethanol and higher n-alkanols

Method used

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  • Novel method for coupled enzymatic reaction-based high-flux detection of ethanol and C3-C6 n-alkanol production capacity of microbes
  • Novel method for coupled enzymatic reaction-based high-flux detection of ethanol and C3-C6 n-alkanol production capacity of microbes
  • Novel method for coupled enzymatic reaction-based high-flux detection of ethanol and C3-C6 n-alkanol production capacity of microbes

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Experimental program
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Effect test

Embodiment 1

[0029] The standard curve of ethanol was determined by the coupling method of alcohol oxidase and catalase. The contents of each substance in Buffer I (200 μL) are: phenol: 6 mmol / ml, EDTA: 75 mg / L, phosphate buffer: pH=7.5, 0.1 mol / L. The contents of each substance in BufferII (100 μL) are: alcohol oxidase: 3000u / L, catalase: 600u / L, 4-antipyrine: 3.5mmol / L, phosphate buffer 0.1mol / L pH= 7.5. Prepare standard ethanol concentrations of 0, 0.1, 0.5, 1.0, 1.5, 2.0, 2.5g / L respectively. Steps: First add 200 μL of Buffer I solution to the 96-well ELISA plate, then add 10 μL of ethanol of different concentrations, and finally add 100 μL of BufferII solution. Incubate for 30min, at 500nm, measure the absorbance value, establish the standard curve of OD500nm absorbance value and ethanol concentration, R 2 =0.999 (accompanying drawing 1), illustrate ethanol concentration and the absorption value at 500nm place to be in significant linear relationship.

Embodiment 2

[0031] Weigh 10g of the hot spring soil sample, and use the culture medium to carry out enrichment culture at 60°C and 200rpm for 24h; dilute the enriched culture flora by 10 5 、10 6 、10 7 times, spread on solid medium, and culture at 60°C for 24 hours. Add 1 mL of culture medium to a 96-deep-well culture plate with a volume of 3 mL per well, inoculate a single colony into it, and culture at 60° C. for 24 h. The 96 deep-well culture plate was centrifuged at 4000 rpm and 4°C for 30 min with an Eppendorf refrigerated centrifuge.

[0032] First add 200 μL of Buffer I solution to the 96-well ELISA plate, then add 10 μL of supernatant, and finally add 100 μL of Buffer II solution. , At 500nm, the absorbance was measured. The ethanol production of 96 strains was calculated according to the ethanol standard curve, and a strain with the highest ethanol production was obtained.

Embodiment 3

[0034] Inoculate the strains obtained from the above screening into the medium, and cultivate them at 200rpm and 60°C for 24 hours; take 10mL of the medium, centrifuge at 10,000rpm for 5min, and use the method of coupling alcohol oxidase and catalase and high-performance liquid phase respectively. The ethanol concentration was measured by the method, and the obtained concentrations were 2.32g / L and 2.39g / L respectively. The two data were basically consistent, and within the error range, it was shown that the method of double-enzyme coupling was reliable for the determination of ethanol.

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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 [0001] The invention belongs to the field of screening, development and utilization of industrial microorganisms, and in particular relates to a high-throughput detection method for detecting ethanol and C3-C6 n-alkanol produced by microorganisms based on a double-enzyme coupling reaction. technical background [0002] Over the past 200 years, the energy system based on fossil fuels such as coal, oil, and natural gas has greatly promoted the development of human society. However, in recent years, due to the excessive exploitation and consumption of oil, natural gas, coal and other energy sources, the world has faced a serious energy crisis, and the international crude oil price has been rising, and the demand for oil is increasing day by day. Since 2010, importing countries have imported 218.45 million tons of crude oil, so finding clean, cheap and renewable alternative energy has become the key to future energy strategies. Fuel ethanol and fuel butanol are ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/30C12Q1/26
Inventor 王钦宏齐显尼涂然孙琳姜丹
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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