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A new method for high-throughput detection of microbial production of isoamyl alcohol based on dual-enzyme coupling

A technology for isoamyl alcohol and microorganisms, which is applied in the field of high-throughput detection of isoamyl alcohol produced by microorganisms, can solve problems such as bottlenecks in screening methods, and achieve the effects of low cost, high efficiency and improved screening speed.

Active Publication Date: 2016-09-14
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Biological methods including metabolic engineering have greatly enriched the resources of engineered strains, but the corresponding screening methods are still a bottleneck

Method used

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  • A new method for high-throughput detection of microbial production of isoamyl alcohol based on dual-enzyme coupling
  • A new method for high-throughput detection of microbial production of isoamyl alcohol based on dual-enzyme coupling
  • A new method for high-throughput detection of microbial production of isoamyl alcohol based on dual-enzyme coupling

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Cloning and expression of isoamyl alcohol oxidase. To measure the content of isoamyl alcohol by coupling isoamyl alcohol oxidase and peroxidase, it is necessary to prepare isoamyl alcohol oxidase.

[0032] (1) The isoamyl alcohol oxidase gene (mreA) is derived from Aspergillus oryzae, and its CICC (China Industrial Microorganism Collection Center) number is 40465. Aspergillus oryzae total RNA was extracted using RNeasy Plant Mini Kit (Qiagen), and RevertAid was used to TM cDNA was reverse transcribed by Premium First Strand cDNA Synthesis Kit #K1651, #K1652 (Fermentas).

[0033] (2) Construction of expression vector pFLDα-mreA. The isoamyl alcohol oxidase gene (mreA) was cloned using Aspergillus oryzae cDNA as a template (Fig. 1). 5'-end primer Primer1: 5'-cggGGTACCatggctgacagctcatcttg-3' (capital letters indicate the Kpn I restriction site); 3'-end primer Primer2: 5'-ctcGGGCCCaacacggcac attctt-3' (capital letters indicate the ApaI restriction site). The primer was...

Embodiment 2

[0039] The standard curve of isoamyl alcohol was determined by coupling isoamyl alcohol oxidase and catalase. The content of each substance in Buffer I is: phenol: 67mM, EDTA: 0.4mM, phosphate buffer: pH=7.5, 0.2mol / L. The content of each substance in BufferII is: peroxidase: 100U / mL, 4-antipyrine: 200mM, phosphate buffer saline pH=7.5, 0.2mol / L. Isoamyl alcohol oxidase crude enzyme liquid: obtained by ultrafiltration and concentration of the fermentation broth after induction and expression of recombinant Pichia pastoris X-33 (pFLDα-mreA) (Example 1), the protein concentration of the crude enzyme liquid was determined by BCA method to be 5.0 mg / mL . Prepare a standard isoamyl alcohol concentration of 1M (in DMSO).

[0040] Steps: Add 60 μL of Buffer I solution, 1M isoamyl alcohol 0, 1.0, 2.0, 3.0, 4.0 μL, 20 μL of Buffer II solution, 20 μL of isoamyl alcohol oxidase crude enzyme solution in a 96 microwell plate, and finally use phosphate buffer Add liquid to a total volume...

Embodiment 3

[0042] Specificity study of a method for the determination of isoamyl alcohol by coupling isoamyl alcohol oxidase and peroxidase. The content of each substance in Buffer I is: phenol: 67mM, EDTA: 0.4mM, phosphate buffer: pH=7.5, 0.2mol / L. The content of each substance in Buffer II is: peroxidase: 100U / mL, 4-antipyrine: 200mM, phosphate buffer solution pH=7.5, 0.2mol / L. Isoamyl alcohol oxidase crude enzyme solution: obtained by ultrafiltration and concentration of the fermentation broth after induction and expression of recombinant Pichia pastoris. The protein concentration in the crude enzyme solution was determined to be 2.0 mg / mL by BCA method. Prepare 2M DMSO solutions of 2-methylbutanol, 3-methylbutanol (isoamyl alcohol), isopropanol, isobutanol, and isohexanol.

[0043] Steps: Add 60 μL of Buffer I solution, 2M 3-methylbutanol, 3-methylbutanol (isoamyl alcohol), 4 μL of isopropanol, isobutanol, and isohexanol, and BufferII solution in a 96-well plate. 20 μL, 20 μL of is...

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Abstract

The invention relates to a new method for high-throughput detection of isoamyl alcohol produced by microorganisms based on a double-enzyme coupling reaction, wherein the isoamyl alcohol oxidase is expressed by recombinant Pichia pastoris. It specifically relates to the coupling of isoamyl alcohol oxidase and peroxidase to react with isoamyl alcohol in a corresponding buffer solution to produce quinone imine, a color-developing substance. The double-enzyme coupling reaction was carried out in a 96-well plate, and the absorbance at 500 nm was read with a microplate reader. The absorbance value at 500nm of the isoamyl alcohol and double-enzyme coupling reaction product is linearly related to the concentration of isoamyl alcohol, and the concentration can be calculated according to the standard curve, and high-throughput detection can be performed using a microplate reader and a 96 microwell plate.

Description

[0001] Inventors: Wang Qinhong, Sun Lin, Tu Ran [0002] Inventor's unit: Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences [0003] Key words: isoamyl alcohol, dual enzyme coupling, high throughput, detection, isoamyl alcohol oxidase [0004] A new method for high-throughput detection of microbial production of isoamyl alcohol based on dual-enzyme coupling technical field [0005] 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 isoamyl alcohol produced by microorganisms based on a double-enzyme coupling reaction. technical background [0006] Due to the depletion of fossil fuels and their destructive impact on the environment, the exploration of sustainable utilization of resources has received more and more attention. In recent years, bioenergy has been gaining more and more favor due to its renewability and less burden o...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/28C12Q1/26
Inventor 王钦宏孙琳涂然
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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