Method for coupling production of sodium pyruvate by co-expressing L-lactate oxidase and hydrogen peroxide

A technology of lactate oxidase and catalase, which is applied in the field of enzyme catalysis, can solve the problems of unsuitability for industrialization, high cost and low yield of enzymatic conversion methods, and achieve the effects of increased yield, simple post-treatment, and high conversion rate

Inactive Publication Date: 2018-11-20
SICHUAN TONGSHENG BIOTECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] Aiming at the problem that the cost of the enzymatic conversion method in the existing pyruvate synthesis method is relatively high or the yield is low and is not suitable for industrialization, the present invention provides a method for co-expressing L-l

Method used

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  • Method for coupling production of sodium pyruvate by co-expressing L-lactate oxidase and hydrogen peroxide
  • Method for coupling production of sodium pyruvate by co-expressing L-lactate oxidase and hydrogen peroxide
  • Method for coupling production of sodium pyruvate by co-expressing L-lactate oxidase and hydrogen peroxide

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Experimental program
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Embodiment 1

[0047] Cultivation of engineering bacteria: Transplant permanent bacteria into the primary medium in a sterile environment, cultivate for 9 hours at a temperature of 37°C and a rotation speed of 220rpm, transfer to the secondary medium after the OD600 reaches 3-4, and then transfer to the secondary medium at a temperature of 37°C. Cultivate at 220rpm for 4-5 hours; then connect it to a fermenter for 37°C cultivation, start feeding when the pH and dissolved oxygen rise, and when the bacterial concentration OD600 reaches 22-28, start to cool down to 30°C, add 0.1mM induction Induced culture with agent IPTG for 12 hours, put in tanks, and collect the bacteria for later use.

[0048] Transformation experiment: 3L 0.2M system transformation was done in a 5L fermenter, the dosage of wet bacteria was 20g / L, the substrate L-sodium lactate was 0.2M, and the content of disodium EDTA was 10mM. Add 60g of the bacteria obtained from fermentation to 1.2L of water, stir well and pour into th...

Embodiment 2

[0051] Cultivation of engineering bacteria: In a sterile environment, transplant permanent bacteria into the primary medium, cultivate at a temperature of 37°C and a rotation speed of 220rpm for 9 hours, and transfer to the secondary medium after the OD600 reaches 3 to 4. Cultivate at 220rpm for 4-5 hours; then connect it to a fermenter for cultivation at 37°C, start feeding when the pH and dissolved oxygen rise, and when the bacterial concentration OD600 reaches 22-28, start to cool down to 30°C, add 0.1mM IPTG to induce culture for 12 Put it in the tank for an hour, and collect the bacteria for later use.

[0052] Transformation experiment: Transform 3L of 0.5M system in a 5L fermenter, the dosage of wet bacteria is 30g / L, the substrate L-sodium lactate is 0.5M, and the content of disodium EDTA is 10mM. Add 90g of wet bacteria to 1.2L of water, stir well and pour into the transformation tank; add 168.8g of L-lactic acid (80%) in total to dilute with water, react with NaOH to...

Embodiment 3

[0055] Cultivation of engineering bacteria: in a sterile environment, inoculate the permanent bacteria in the primary medium, cultivate at a temperature of 37°C and a rotational speed of 220rpm for 9 hours, and transfer to the secondary medium after the OD600 reaches 3-4, and incubate at a temperature of 37°C with a rotational speed of 220 Cultivate at 220rpm for 4-5 hours; then connect to a fermenter for cultivation at 37°C, start feeding when the pH and dissolved oxygen rise, and when the bacterial concentration OD600 reaches 22-28, start to cool down to 30°C, add 0.1mM IPTG to induce cultivation for 12 hours Put it in a tank and collect the bacteria for later use.

[0056] Transformation experiment: Transform 6L of 1.0M system in a 10L fermenter, the dosage of wet bacteria is 40g / L, the substrate L-sodium lactate is 1.0M, and the content of disodium EDTA is 10mM. Add 240g of wet bacteria to 2.4L of water, stir well and pour into the transformation tank; add 675g of L-lactic...

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Abstract

The invention belongs to the technical field of enzymatic catalysis, and particularly relates to a method for coupling production of sodium pyruvate by co-expressing L-lactate oxidase and hydrogen peroxide. Aiming at the problem that an enzymatic conversion method in an existing synthetic method of pyruvic acid is higher in cost and further is unsuitable for industrialization, the method is characterized by comprising the following steps: firstly, constructing a coexpression vector of the L-lactate oxidase and the hydrogen peroxide and transferring the coexpression vector into Escherichia coli; secondly, carrying out high-density fermentation on the Escherichia coli obtained in the first step to obtain L-lactate oxidase and hydrogen peroxide coexpressed Escherichia coli engineering bacteria; thirdly, putting a wet cell mass of the Escherichia coli engineering bacteria obtained in the second step into an L-sodium lactate solution, introducing sterile air, and stirring and transforming to obtain transformed liquid; and fourthly, carrying out microfiltration and ultrafiltration on the transformed liquid obtained in the third step to remove thalli and protein, and carrying out impurityremoval and alcohol precipitation to obtain the sodium pyruvate. The method disclosed by the invention is suitable for industrial production of the sodium pyruvate.

Description

technical field [0001] The invention belongs to the technical field of enzyme catalysis, and in particular relates to a method for co-expressing L-lactate oxidase and catalase to couple and produce sodium pyruvate. Background technique [0002] Pyruvate, also known as pyrogluconate, is the initial substrate of the tricarboxylic acid cycle and widely exists in organisms. In chemical synthesis, pyruvic acid is a very important intermediate and is in great demand. Pyruvate is the precursor for the biosynthesis of L-dopa, L-tyrosine, and L-tryptophan, and it is also the initial substrate for the synthesis of pesticides. At present, the production of pyruvate mainly adopts three methods: chemical synthesis, fermentation accumulation and enzymatic conversion. [0003] 1. The chemical synthesis method follows the dehydration and decarboxylation of tartaric acid to generate pyruvic acid reported by Howard and Fraser in 1932. It has a large pollution and a relatively low yield (0.2...

Claims

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Application Information

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IPC IPC(8): C12P7/40C12N15/70C12N15/66C12N15/53C12R1/19
CPCC12N9/0006C12N9/0065C12N15/66C12N15/70C12P7/40C12Y101/01027C12Y111/01006
Inventor 王苓李晚军刘超曾帅刘鑫杨顺楷陈纹锐
Owner SICHUAN TONGSHENG BIOTECH
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