Novel high-throughput detection method for microbial production for dibasic acid based on coupled enzymatic reaction

A dibasic acid and microbial technology, applied in biochemical equipment and methods, microbial determination/testing, etc., can solve the problems of retention time offset, low sensitivity, expensive NADH, etc., and achieve increased screening speed, high sensitivity, and low cost effect

Inactive Publication Date: 2014-02-12
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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Problems solved by technology

[0004] However, the traditional methods for qualitative or quantitative detection of dibasic acids are very limited. Taking succinic acid as an example, the main detection methods include liquid chromatography, gas chromatography, reversed-phase high performance liquid chromatography and kit detection.
The operation of liquid chromatography is cumbersome and the sensitivity is low. If there are many samples to be detected, there will also be problems such as offset of retention time.
The use of kits to detect succinic acid is not only expensive, but also requires strict sample purity and will be interfered by other substances
The detection of the kit needs to rely on NADH, but NADH is quite expensive, and the absorbance value needs to be measured at 340nm, so a UV detector is required, which increases the cost of detection

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  • Novel high-throughput detection method for microbial production for dibasic acid based on coupled enzymatic reaction
  • Novel high-throughput detection method for microbial production for dibasic acid based on coupled enzymatic reaction
  • Novel high-throughput detection method for microbial production for dibasic acid based on coupled enzymatic reaction

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Embodiment 1

[0028] Dual-enzyme-coupled fluorometric method compared to chemichromogenic method.

[0029] (1) Fluorescence method. Reagent A: AmplexUltraRed: 0.1 mM, Peroxidase: 9 U / mL, PBS: pH 7.4. Hydrogen peroxide solution: 0.1 mM. Steps: Add different concentrations of H in sequence on a 96-well black microplate 2 o 2 (PBS dilution) 50uL, reagent A 50μL, the total volume is 100μL, measure Ex / Em490 / 585 (nm) fluorescence value. Establish Ex / Em 490 / 585(nm) fluorescence value and H 2 o 2 Concentration standard curve (attached figure 1 ), R 2 =0.992, indicating that the dual-enzyme-coupled fluorescence method can detect μM level of H 2 o 2 .

[0030] (2) Chemical chromogenic method. The content of each substance in Buffer I is: phenol: 6mmol / mL, EDTA: 75mg / L, phosphate buffer: pH=7.5, 0.1mol / L. The content of each substance in BufferII is: catalase: 600U / L, 4-antipyrine: 3.5mmol / L, 0.1mol / L phosphate buffer solution pH=7.5. Steps: Add different concentrations of H to the 96-wel...

Embodiment 2

[0032]The method of coupling fumarate reductase and peroxidase is used to measure the change of the fluorescence value of succinic acid with time and the determination of the measurement time. Reagent A: AmplexUltraRed: 0.1 mM, Peroxidase: 9 U / mL, PBS: pH 7.4. Fumarate reductase crude enzyme solution: recombinant Escherichia coli E. coli BL21 (pET-30a-frd) was induced to express and treated with protein extraction reagent to obtain crude enzyme solution. Prepare 1 mM succinic acid solution. Steps: Add 20 μL of PBS, 10 μL of 1 mM succinic acid (diluted in PBS), 50 μL of reagent A, and 20 μL of crude fumarate reductase enzyme solution to a 96-well black microwell plate, with a total volume of 100 μL. The control group did not add succinic acid. Set the temperature of the microplate reader to 37°C, and read the Ex / Em 490 / 585 (nm) fluorescence value every 5 minutes for 0-30 minutes. From the time change graph (attached Figure 4 ), it can be seen that the crude enzyme solution...

Embodiment 3

[0034] The standard curve of succinic acid was determined by the coupling method of fumarate reductase and peroxidase. Reagent A: AmplexUltraRed: 0.1 mM, Peroxidase: 9 U / mL, PBS: pH 7.4. Fumarate reductase crude enzyme solution: recombinant Escherichia coli E. coli BL21 (pET-30a-frd) was induced to express and treated with protein extraction reagent to obtain crude enzyme solution. Prepare 1 mM succinic acid solution. Steps: 30 μL of different concentrations of succinic acid (diluted in PBS), 50 μL of reagent A, and 20 μL of crude fumarate reductase enzyme solution were sequentially added to a 96-well black microwell plate, with a total volume of 100 μL. Incubate at 37°C for 30 minutes, and read the fluorescence value of Ex / Em 490 / 585 (nm). Establish the standard curve of Ex / Em 490 / 585 (nm) fluorescence value and succinic acid concentration (attachment Figure 5 ), R 2 =0.998, indicating that the concentration of succinic acid has a significant linear relationship with the...

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Abstract

The invention relates to a novel high-throughput detection method for microbial production for dibasic acid based on a coupled enzymatic reaction, and in particular to a method for generating a fluorescent substrate Resorufin by virtue of coupling between dibasic acid oxidase and peroxidase, and reaction with dibasic acid in corresponding buffering solution. According to the method, the coupled enzymatic reaction is performed in a 96-mesh black microporous plate, and a fluorescent value at a place of Ex/Em 490/585 (nm) is read by an enzymatic analyzer; and the fluorescent value of the product generated by the coupled reaction between the dibasic acid and the both enzymes at the place of Ex/Em 490/585 (nm) forms a linear relationship with the concentration of the product, the concentration can be calculated according to a standard curve, and high-throughput detection is performed by the enzymatic analyzer and the 96-mesh black microporous plate.

Description

technical field [0001] The invention belongs to the field of screening, development and utilization of industrial microorganisms, in particular to a high-throughput detection method for detecting dibasic acids produced by microorganisms based on a double-enzyme coupling reaction. technical background [0002] Screening from nature and mutation evolution in the laboratory has always been an important way to discover and innovate industrial microbial strains, and is the foundation of the industrial biotechnology industry. Metabolic engineering has greatly facilitated the improvement of engineered strains of various compounds used in industry. Especially in recent decades, metabolic engineering has entered a new stage - systemic metabolic engineering. The organic integration of systems metabolic engineering with systems biology with global concepts, synthetic biology with fine design capabilities, and evolutionary engineering with rational-random mutations makes metabolic engi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/28C12Q1/26
Inventor 王钦宏孙琳涂然齐显尼姜丹马延和
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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