Method for expressing and purifying recombinant ethanol oxidase in pichia pastoris

A technology of alcohol oxidase and alcohol oxidase protein, which is applied in the direction of oxidoreductase, biochemical equipment and methods, recombinant DNA technology, etc., can solve the problem of enzyme inactivity, etc., and achieve increased expression, optimized fermentation conditions, and enzyme purity. high effect

Inactive Publication Date: 2012-07-04
ZHEJIANG DEQING HUINING BIOTECH
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Problems solved by technology

[0005] In terms of recombinant expression, the heterologous expression of alcohol oxidase has been reported. For example, the recombinant expression of alcohol oxidase has been carried out in Saccharomyces cerevisiae and Escherichia coli. Although the expression is successful, the expressed enzyme has no activity.

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  • Method for expressing and purifying recombinant ethanol oxidase in pichia pastoris
  • Method for expressing and purifying recombinant ethanol oxidase in pichia pastoris
  • Method for expressing and purifying recombinant ethanol oxidase in pichia pastoris

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Embodiment Construction

[0032] The present invention is further described by steps below:

[0033] Step 1, Pichia pastoris genome extraction method:

[0034] 1) Inoculate Pichia pastoris GS115 to 5ml yeast extract peptone glucose (YPD) medium, culture at 30°C for 16-18 hours, take 1.5ml of yeast culture solution, and centrifuge at 5000 rpm for 5-10 minutes at room temperature to collect the bacteria ;

[0035] 2) The cells were washed once with sterile water and centrifuged at 5000 rpm for 5 minutes; the cells were resuspended in 200 μL TE buffer, and 30 μL 10 mg / ml lysozyme was added to incubate at 37°C for 1 hour; 100 μL 2% dodecyl Mix sodium sulfate (SDS), freeze at -20°C, then shake at room temperature to dissolve, repeat 3 times; add 150 μL 5mol / L potassium acetate with pH 8.9 to the suspension, mix gently, and then centrifuge at 10,000 rpm for 5 minute;

[0036] 3) Transfer the supernatant to a new 1.5ml centrifuge tube, add 2 times the volume of ethanol, mix gently, leave at room temperatur...

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Abstract

The invention discloses a method for expressing and purifying recombinant ethanol oxidase rAOX. According to the method, an ethanol oxidase-poly-L-histidine expression vector pPIC9K-AOX-HIS is constructed first by a molecular biological technique, then the expression vector pPIC9K-AOX-HIS is linearized and transferred to pichia pastoris GS115, and thus, pichia pastoris engineering bacteria capable of expressing active recombinant ethanol oxidase are obtained, and part of recombinant ethanol oxidase can be secreted and expressed. Through optimization of the expression conditions of recombinant ethanol oxidase, the ethanol oxidase produced by the engineering bacteria may reach 1,862U / L. Meanwhile, the secretion and expression of the ethanol oxidase and the introduction of a poly-L-histidine tag simplify the purification process of the recombinant ethanol oxidase. According to the result of tests, the recombinant ethanol oxidase expressed by the engineering bacteria has much higher thermostability than pichia pastoris wide type ethanol oxidase. The operation in the whole process of the method is simple, so the method is suitable for industrial production and can create certain economic benefit.

Description

technical field [0001] The invention relates to a method for expressing and purifying recombinant alcohol oxidase. More specifically, it relates to a method for expressing and purifying recombinant alcohol oxidase in Pichia pastoris. Background technique [0002] Alcohol oxidase is an important enzyme in methanol metabolism, which can catalyze methanol and other lower alcohols and oxidize them to generate their corresponding aldehydes and hydrogen peroxide. The active form of alcohol oxidase is a homologous octamer with a molecular weight of 600kDa, and is located in the peroxisome of the cell. Each subunit of alcohol oxidase contains a flavin adenine dinucleotide coenzyme. The subunit of alcohol oxidase is formed in the periplasm, and after binding with flavin adenine dinucleotide, enters the peroxisome to assemble octamer. [0003] Alcohol oxidase can effectively catalyze methanol and other lower alcohols by consuming oxygen. In fact, many ethanol detectors based on oxy...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N15/81C12N9/04
Inventor 徐志南刘云平朱建忠
Owner ZHEJIANG DEQING HUINING BIOTECH
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