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Novel method for producing isoamyl alcohol based on double-enzyme coupling high flux detection microbe

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, low cost and high throughput

Active Publication Date: 2014-10-15
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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  • Novel method for producing isoamyl alcohol based on double-enzyme coupling high flux detection microbe
  • Novel method for producing isoamyl alcohol based on double-enzyme coupling high flux detection microbe
  • Novel method for producing isoamyl alcohol based on double-enzyme coupling high flux detection microbe

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] 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.

[0028] (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).

[0029] (2) Construction of expression vector pFLDα-mreA. Isoamyl alcohol oxidase gene (mreA) was cloned using Aspergillus oryzae cDNA as a template (attached figure 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 p...

Embodiment 2

[0035] The standard curve of isoamyl alcohol was determined by the coupling method of 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).

[0036] 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 ...

Embodiment 3

[0038]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 liquid: obtained by ultrafiltration and concentration of the fermentation broth after induction and expression of recombinant Pichia pastoris. The protein concentration in the crude enzyme liquid was determined by BCA method to be 2.0 mg / mL. Prepare 2M DMSO solutions of 2-methylbutanol, 3-methylbutanol (isoamyl alcohol), isopropanol, isobutanol, and isohexanol.

[0039] 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 to a 96 microwell plate in sequence 20 μL, ...

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Abstract

The invention relates to a novel method for producing isoamyl alcohol based on a high flux detection microbe of a double-enzyme coupling reaction. Isoamyl alcohol oxidase is obtained through expression of recombinant pichia pastoris. Specifically speaking, isoamyl alcohol oxidase is coupled with peroxidase, reactions with isoamyl alcohol are carried out in corresponding buffer solutions, and the chromogenic substance quinone-imine is produced; the double-enzyme coupling reaction is carried out in a 96 microwell plate, and a microplate reader is employed to read the light absorption value at 500 nm; since a linear relation is formed between the light absorption value of the resultant of the coupling reaction of isoamyl alcohol with the double enzymes at 500 nm and the concentration of isoamyl alcohol, the concentration can be calculated according to a standard curve, and the microplate reader and the 96 microwell plate are used for high flux detection.

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 isoamyl alcohol produced by microorganisms based on a double-enzyme coupling reaction. technical background [0002] 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 on the environment. The diversity of microorganisms, the rapid development of biotechnology, the continuous innovation of research methods and the upgrading of instruments have greatly promoted the development and utilization of bioenergy. Taking bioethanol as an example, the production of bioethanol in the United States reached 9.2 billion gallons (about 35 billion liters) in 2008. Ho...

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
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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